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Viruses
Volume 7
Issue 9
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Viruses, Volume 7, Issue 9 (September 2015) – 18 articles

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690 KiB  
Review
Interferon-Free Hepatitis C Treatment before and after Liver Transplantation: The Role of HCV Drug Resistance
by Bruno Roche, Audrey Coilly, Anne-Marie Roque-Afonso and Didier Samuel
Viruses 2015, 7(9), 5155-5168; https://doi.org/10.3390/v7092864 - 23 Sep 2015
Cited by 19 | Viewed by 7001
Abstract
Hepatitis C virus (HCV) infection is one of the leading causes of end-stage liver disease and the main indication for liver transplantation (LT) in most countries. All patients who undergo LT with detectable serum HCV RNA experience graft reinfection progressing to cirrhosis within [...] Read more.
Hepatitis C virus (HCV) infection is one of the leading causes of end-stage liver disease and the main indication for liver transplantation (LT) in most countries. All patients who undergo LT with detectable serum HCV RNA experience graft reinfection progressing to cirrhosis within five years in 20% to 30% of them. Obtaining a sustained virological response (SVR) greatly improves overall and graft survival. Until 2011, standard antiviral therapy using PEGylated interferon (PEG-IFN) and ribavirin (RBV) was the only effective therapy, with an SVR rate around 30% in this setting. For patients infected with genotype 1, first generation NS3/4A protease inhibitors (PIs), boceprevir (BOC) or telaprevir (TVR), associated with PEG-IFN and RBV for 48 weeks have increased the SVR rates to 60% in non-transplant patients. However, tolerability and drug-drug interactions with calcineurin inhibitors (CNI) are both limiting factors of their use in the liver transplant setting. Over recent years, the efficacy of antiviral C therapy has improved dramatically using new direct-acting antiviral (DAA) agents without PEG-IFN and/or RBV, leading to SVR rates over 90% in non-transplant patients. Results available for transplant patients showed a better efficacy and tolerability and less drug-drug interactions than with first wave PIs. However, some infrequent cases of viral resistance have been reported using PIs or NS5A inhibitors pre- or post-LT that can lead to difficulties in the management of these patients. Full article
(This article belongs to the Special Issue HCV Drug Resistance)
733 KiB  
Review
Tight Junctions Go Viral!
by Jesús M. Torres-Flores and Carlos F. Arias
Viruses 2015, 7(9), 5145-5154; https://doi.org/10.3390/v7092865 - 23 Sep 2015
Cited by 75 | Viewed by 12923
Abstract
Tight junctions (TJs) are highly specialized membrane domains involved in many important cellular processes such as the regulation of the passage of ions and macromolecules across the paracellular space and the establishment of cell polarity in epithelial cells. Over the past few years [...] Read more.
Tight junctions (TJs) are highly specialized membrane domains involved in many important cellular processes such as the regulation of the passage of ions and macromolecules across the paracellular space and the establishment of cell polarity in epithelial cells. Over the past few years there has been increasing evidence that different components of the TJs can be hijacked by viruses in order to complete their infectious cycle. Viruses from at least nine different families of DNA and RNA viruses have been reported to use TJ proteins in their benefit. For example, TJ proteins such as JAM-A or some members of the claudin family of proteins are used by members of the Reoviridae family and hepatitis C virus as receptors or co-receptors during their entry into their host cells. Reovirus, in addition, takes advantage of the TJ protein Junction Adhesion Molecule-A (JAM-A) to achieve its hematogenous dissemination. Some other viruses are capable of regulating the expression or the localization of TJ proteins to induce cell transformation or to improve the efficiency of their exit process. This review encompasses the importance of TJs for viral entry, replication, dissemination, and egress, and makes a clear statement of the importance of studying these proteins to gain a better understanding of the replication strategies used by viruses that infect epithelial and/or endothelial cells. Full article
(This article belongs to the Section Animal Viruses)
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<p>Tight junction proteins and virus replication. Occludin is required by hepatitis C virus, coxsackie B virus, and rotavirus during entry into their host cell. West Nile virus is capable of opening tight junctions by impairing occludin in order to achieve its hematogenous dissemination. Claudins-1, -6, and -9 are used by hepatitis C virus as entry factors. Claudin-1 is also an entry factor for dengue virus, while West Nile virus targets claudin-1 (CLDN-1) to open tight junctions to achieve hematogenous dissemination. Reovirus and feline calicivirus both use JAM-A as a receptor during their entry process, while rotavirus needs JAM-A as a co-receptor. Adenoviruses and coxsackievirus B use CAR as a receptor during their entry. Several proteins of the cytoplasmic plaque that forms tight junctions are involved in viral replication. Rotaviruses require the organization of the plaque provided by the protein zonula occludens protein-1 (ZO-1) for their entry, while other viruses, like influenza, severe acute respiratory syndrome (SARS), and West Nile target this protein to disrupt tight junctions to spread and disseminate. Other plaque proteins, like the multi-PDZ domain protein-1, -2, and -3 (MUPP1, MUPP2, MUPP3), membrane-associated guanylate kinase, WW, and PDZ domain-containing protein-1 (MAGI-1), PALS1-associated TJ protein (PATJ), and zonula occludens protein-2 (ZO-2) are hijacked by adenoviruses and other respiratory viruses, like influenza virus and SARS coronavirus, to open the tight junctions and efficiently exit the airway epithelia. Finally, tight junction proteins are also involved in carcinogenesis, PATJ and MUPP1 are targeted by human papillomaviruses to alter cell polarity, an event that is capable of inducing carcinogenesis in epithelial cells.</p> Full article ">
2331 KiB  
Article
Preventive Activity against Influenza (H1N1) Virus by Intranasally Delivered RNA-Hydrolyzing Antibody in Respiratory Epithelial Cells of Mice
by Seungchan Cho, Ha-Na Youn, Phuong Mai Hoang, Sungrae Cho, Kee-Eun Kim, Eui-Joon Kil, Gunsup Lee, Mun-Ju Cho, Juhyun Hong, Sung-June Byun, Chang-Seon Song and Sukchan Lee
Viruses 2015, 7(9), 5133-5144; https://doi.org/10.3390/v7092863 - 21 Sep 2015
Cited by 11 | Viewed by 6623
Abstract
The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic [...] Read more.
The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 μg/day) for five days prior to infection demonstrated an antiviral activity (70% survival) against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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<p>Direct catalytic activity of 3D8 scFv against H1N1 influenza virus. Madin-Darby Canine Kidney epithelical cells (MDCK cells) were infected with 200 μL of 10<sup>3</sup> EID<sub>50</sub> influenza virus for 4 h and then incubated for 24 h in serum-free medium with trypsin (1 μg/mL). (<b>A</b>) The cytopathic effects were examined by microscopy. Magnification 100×. The arrows indicated the cytopathic effects on host cells caused by H1N1 infection; (<b>B</b>) Transcripts of hemagglutinin and neuraminidase were measured by qRT-PCR and normalized by against GAPDH cDNA using the 2<sup>−ΔΔ<span class="html-italic">C</span>t</sup> method. Data are shown as mean ± S.E.M of triplicate samples from three independent experiments. Data are mean ± standard error. <b>***</b> Significantly different from 3D8 scFv/H1N1 group at <span class="html-italic">p</span> &lt; 0.001 (one-way analysis of variance and Tukey’s <span class="html-italic">post hoc</span> <span class="html-italic">t</span>-test); (<b>C</b>) The RNA transcript of hemagglutinin was incubated with 3D8 scFv purified protein for 1 h; (<b>D</b>) Reactions were terminated at 10, 20, 30, 40, 50 and 60 min and analyzed by electrophoresis.</p> Full article ">Figure 2
<p>Antiviral effects of intranasally administrated 3D8 scFv on survival and body weight. (<b>A</b>) BALB/c mice were treated intranasally with 3D8 scFv protein (50 μg/mouse) for 3 or 5 days before infection with A/NWS/33; (<b>B</b>,<b>C</b>) Mice were monitored daily for 14 days to determine the rate of survival (<b>B</b>) and changes in body weight (<b>C</b>). Control group, <span class="html-italic">n</span> = 10; positive control group, <span class="html-italic">n</span> = 10; treatment groups, <span class="html-italic">n</span> = 10. Asterisks indicate significant differences (<b>*</b> <span class="html-italic">p</span> &lt; 0.05, <b>**</b> <span class="html-italic">p</span> &lt; 0.01) compared with the positive (H1N1) control group (Fisher’s exact test).</p> Full article ">Figure 3
<p>Preventative antiviral effects of 3D8 scFv protein in the lung. Emulsion samples were extracted from lung tissue after virus infection. (<b>A</b>) The viral titer was measured by TCID<sub>50</sub> assay; (<b>B</b>) Viral replication was analyzed by qRT-PCR; (<b>C</b>) To confirm viral protein expression, H1N1 HA protein was detected by confocal microscopy using antibodies specific for HA and GAPDH. Asterisks indicate significant differences (<b>*</b> <span class="html-italic">p</span> &lt; 0.05, <b>**</b> <span class="html-italic">p</span> &lt; 0.01) versus the positive (H1N1) control group (one-way analysis of variance and Tukey’s <span class="html-italic">post hoc t</span>-test); (<b>D</b>) Photomicrographs of lung sections in H1N1-infected mice treated with 3D8 scFv. Lung sections from mice at 3 days post challenge were stained with H&amp;E. Uninfected lungs without treatment [panels (<b>a</b>,<b>d</b>), alveoli; (<b>g</b>,<b>j</b>), bronchiole]; infected lung without treatment [panels (<b>b</b>,<b>e</b>), alveoli; (<b>h</b>,<b>k</b>), bronchiole]; and infected lung treated with 3D8 scFv [panels (<b>c</b>,<b>f</b>), alveoli; (<b>i</b>,<b>l</b>), bronchiole].</p> Full article ">Figure 4
<p>Penetration of 3D8 scFv into the epithelium of the nasal mucosa and analysis of cytokine and chemokine expression. (<b>A</b>) The presence of 3D8 scFv in the epithelium of the nasal mucosa of the lung was detected by immunohistochemistry. Lung tissues were stained with anti-3D8 scFv polyclonal Ab and visualized using a TRITC-conjugated anti-rabbit secondary Ab and fluorescence microscopy. Mice were treated with or without 3D8 scFv for 5 days and then challenged. After virus challenge, lung samples were extracted from each group on days 3 and 6 p.i.; (<b>B</b>) mRNA expression of the indicated cytokines and chemokines was measured by qRT-PCR with primers against IFN-β, IFN-γ, or GADPH.</p> Full article ">
1024 KiB  
Review
Conformational Masking and Receptor-Dependent Unmasking of Highly Conserved Env Epitopes Recognized by Non-Neutralizing Antibodies That Mediate Potent ADCC against HIV-1
by George K. Lewis, Andrés Finzi, Anthony L. DeVico and Marzena Pazgier
Viruses 2015, 7(9), 5115-5132; https://doi.org/10.3390/v7092856 - 18 Sep 2015
Cited by 38 | Viewed by 8725
Abstract
The mechanism of antibody-mediated protection is a major focus of HIV-1 vaccine development and a significant issue in the control of viremia. Virus neutralization, Fc-mediated effector function, or both, are major mechanisms of antibody-mediated protection against HIV-1, although other mechanisms, such as virus [...] Read more.
The mechanism of antibody-mediated protection is a major focus of HIV-1 vaccine development and a significant issue in the control of viremia. Virus neutralization, Fc-mediated effector function, or both, are major mechanisms of antibody-mediated protection against HIV-1, although other mechanisms, such as virus aggregation, are known. The interplay between virus neutralization and Fc-mediated effector function in protection against HIV-1 is complex and only partially understood. Passive immunization studies using potent broadly neutralizing antibodies (bnAbs) show that both neutralization and Fc-mediated effector function provides the widest dynamic range of protection; however, a vaccine to elicit these responses remains elusive. By contrast, active immunization studies in both humans and non-human primates using HIV-1 vaccine candidates suggest that weakly neutralizing or non-neutralizing antibodies can protect by Fc-mediated effector function, albeit with a much lower dynamic range seen for passive immunization with bnAbs. HIV-1 has evolved mechanisms to evade each type of antibody-mediated protection that must be countered by a successful AIDS vaccine. Overcoming the hurdles required to elicit bnAbs has become a major focus of HIV-1 vaccine development. Here, we discuss a less studied problem, the structural basis of protection (and its evasion) by antibodies that protect only by potent Fc-mediated effector function. Full article
(This article belongs to the Special Issue Viral Glycoprotein Structure)
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<p>Structure of the A32 sub-region of Epitope Cluster A. Panel (<b>a</b>) depicts the mobile layers of gp120 as defined in [<a href="#B95-viruses-07-02856" class="html-bibr">95</a>,<a href="#B96-viruses-07-02856" class="html-bibr">96</a>] where layer 1 is yellow, layer 2 is cyan, the 7-stranded β-sandwich is red, the gp120 outer domain is bronze and the gp120 inner domain is gray; Panel (<b>b</b>) shows epitope contacts for mAb N5-i5 from [<a href="#B93-viruses-07-02856" class="html-bibr">93</a>] rendered as cpk structures and colored according to the mobile layers in panel <b>a</b>; Panel (<b>c</b>) shows epitope contacts for mAb 2.2c from [<a href="#B93-viruses-07-02856" class="html-bibr">93</a>] rendered as cpk structures and colored according to the mobile layers in panel <b>a</b>; Panel (<b>d</b>) shows the relationships between epitope contact residues for mAb N5-i5 binding to gp120<sub>93TH057</sub> and 2.2c binding to gp120<sub>89.6P</sub> from [<a href="#B93-viruses-07-02856" class="html-bibr">93</a>]. The coloring scheme for mobile layers 1 and 2 as well as the 7-stranded β-sandwich are the same as for panel <b>a</b>.</p> Full article ">Figure 2
<p>Epitope binding angle determines the orientation of IgG1 toward or away from Fc-receptors on the effector cell membrane. Panel (<b>a</b>) shows the predicted orientation of the CH2 domain of N5-i5 IgG1 when it is bound to a gp120-CD4 complex on the target cell membrane facilitating its recognition by an FcγR on the effector cell membrane (from [<a href="#B93-viruses-07-02856" class="html-bibr">93</a>]). Four domain cell surface CD4 was generated using PDB:1WIO (ectodomain) and PDB:2KLU (transmembrane and cytoplasmic domain). The N5-i5 complex with gp120 used PDB:4H8W and 2.2c complex with gp120 used PDB:4R4F. The 7S IgG1 structure was from PDB:1GY and the human FcγR3a-human IgG1 Fc complex structure was from PDB:1E4K. Cell surface CD4 is colored as steel, gp120 as beige, IgG1 heavy chain as red, light chain as blue, and FcγR3a is yellow. The parallelogram represents the target cell membrane. The figure was constructed using ICMPro, Molsoft, LLC, La Jolla, CA, USA; Panel (<b>b</b>) shows the predicted orientation of the CH2 domain of 2.2c IgG1 when it is bound to a gp120-CD4 complex on the target cell membrane disfavoring its recognition by an FcγR on the effector cell membrane (from [<a href="#B93-viruses-07-02856" class="html-bibr">93</a>]).</p> Full article ">Figure 3
<p>Structure of the C11 sub-region of Epitope Cluster A. Panel (<b>a</b>) shows the mobile layers of gp120 as defined in [<a href="#B95-viruses-07-02856" class="html-bibr">95</a>,<a href="#B96-viruses-07-02856" class="html-bibr">96</a>] where layer 1 is yellow, layer 2 is cyan, and the 7-stranded β-sandwich is red, the gp120 outer domain is bronze and the gp120 inner domain is gray; Panel (<b>b</b>) shows residues 45, 88, 491,493, and 495 that are putative contact sites for C11 [<a href="#B96-viruses-07-02856" class="html-bibr">96</a>,<a href="#B99-viruses-07-02856" class="html-bibr">99</a>]. The putative C11 contact residues map into the 7-stranded β-sandwich and the C-Terminal extension of the gp120.</p> Full article ">Figure 4
<p>Epitope Cluster A maps into the gp41 docking site for gp120 in Env trimer mimetic structures. The leftmost structure is the soluble SOSIP Env timer mimetic from PDB:4TVP [<a href="#B95-viruses-07-02856" class="html-bibr">95</a>] where gp120 is red and gp41 is gray. The middle figure is a gp41 monomer from PDB:4TVP in gray. The gp41 interactive face comprised of elements from mobile layer 1 (yellow), mobile layer 2 (cyan), the 7-stranded β-sandwich (green), and the N- and C-Terminal extensions (red) of monomeric gp120 shown as ribbon diagrams. The upper rightmost figure is the same as the middle figure except with the N5-i5 and C11 contact residues rendered as cpk structures. The lower rightmost figure is the same as the upper rightmost figure rotated approximately 90°. Note that the N5-i5 contact residues are in mobile layers 1 and 2 (yellow and cyan), whereas the C11 contact residues are in the 7-stranded β-sandwich (green). The viral membrane would be at the bottom of each structure.</p> Full article ">
2568 KiB  
Review
Tegument Assembly and Secondary Envelopment of Alphaherpesviruses
by Danielle J. Owen, Colin M. Crump and Stephen C. Graham
Viruses 2015, 7(9), 5084-5114; https://doi.org/10.3390/v7092861 - 18 Sep 2015
Cited by 151 | Viewed by 23249
Abstract
Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument [...] Read more.
Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. Full article
(This article belongs to the Special Issue Viruses and Exosomes)
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<p>Neuronal trafficking during entry and egress. Alphaherpesviruses establish latent infection in the nuclei of peripheral ganglia following retrograde transport of capsids along microtubules. Reactivation results in the production of new virions that undergo anterograde trafficking back to peripheral tissues. The assembly state of viral particles prior to anterograde axonal transport is disputed and two models have been proposed: the “married model” predicts that virions are assembled in the cell body and trafficked within vesicles; the “separate model” predicts that capsids and secondary-envelopment membranes are trafficked separately with final virion assembly occurring at or near the sites of egress. Minus-end directed transport to the cell body along microtubules is driven by dynein while kinesins drive plus-end directed transport to the cell periphery. The movement of viral particles along axons during entry and egress is bidirectional and saltatory suggesting that both dynein and kinesin motor proteins may be involved. How the net direction of transport during entry and egress is determined is currently unknown.</p> Full article ">Figure 2
<p>Maturation and egress of herpesviruses. Replication of the viral genome and encapsidation occurs in the nucleus. Once assembled, capsids interact with the inner nuclear membrane and bud into the perinuclear space where they form primary enveloped particles. The primary envelope is then lost upon fusion with the outer nuclear membrane and unenveloped capsids are released into the cytoplasm. Cytoplasmic capsids acquire tegument proteins and their membrane by budding into specialised vesicles, probably derived from endosomes or the trans-Golgi network (TGN), that are studded with viral glycoproteins and outer tegument proteins. The secondary envelopment step also provides a transport vesicle that later fuses with the plasma membrane (PM) to release enveloped virions from the cell.</p> Full article ">Figure 3
<p>Conserved alphaherpesvirus tegument proteins (blue) link the capsid (yellow) to the glycoproteins and envelope proteins (green) embedded in the virion lipid bilayer envelope (grey). Tegument assembles via a dense network of protein:protein interactions: solid lines indicate interactions demonstrated in HSV and dashed lines show interactions demonstrated for PrV. Some tegument proteins associate directly with the envelope via post-translational modifications conferring lipophilic palmitoyl (red) or myristoyl (purple) groups. The proteins that comprise the portal vertex associated tegument (PVAT) are currently undefined.</p> Full article ">Figure 4
<p>Protein pUL36 extends from capsid vertices and interacts with the capsid vertex-specific component (CVSC). (Top inset) The extended N-terminal region of pUL36 interacts with pUL37 and pUL48. For clarity pUL36 and pUL37 are not drawn to scale. (Bottom inset) Recent studies of HSV, PrV and KSHV [<a href="#B107-viruses-07-02861" class="html-bibr">107</a>,<a href="#B135-viruses-07-02861" class="html-bibr">135</a>,<a href="#B145-viruses-07-02861" class="html-bibr">145</a>] suggest that CVSC component pUL25 lies over the penton vertex, pUL17 lies above the penton proximal pUL18-pUL38 triplex, a C-terminal region of pUL36 contributes to the CVSC density, and that pUL36 is essential for CVSC formation.</p> Full article ">Figure 5
<p>Proteins pUL11, pUL16 and pUL21 may form a tripartite complex that binds gE. The C-terminal domain of pUL16 inhibits its ability to co-localise with pUL11 and gE, co-localization of pUL16 with pUL11 is enhanced in the presence of pUL21, and the presence of pUL11 promotes co-localization of pUL16 and gE [<a href="#B101-viruses-07-02861" class="html-bibr">101</a>]. An alternative model is that pUL16 acts as a molecular chaperone, promoting the correct folding of pUL11, pUL21 and/or gE.</p> Full article ">
1199 KiB  
Review
Exosome Biogenesis, Regulation, and Function in Viral Infection
by Marta Alenquer and Maria João Amorim
Viruses 2015, 7(9), 5066-5083; https://doi.org/10.3390/v7092862 - 17 Sep 2015
Cited by 290 | Viewed by 21676
Abstract
Exosomes are extracellular vesicles released upon fusion of multivesicular bodies(MVBs) with the cellular plasma membrane. They originate as intraluminal vesicles (ILVs) duringthe process of MVB formation. Exosomes were shown to contain selectively sorted functionalproteins, lipids, and RNAs, mediating cell-to-cell communications and hence playing [...] Read more.
Exosomes are extracellular vesicles released upon fusion of multivesicular bodies(MVBs) with the cellular plasma membrane. They originate as intraluminal vesicles (ILVs) duringthe process of MVB formation. Exosomes were shown to contain selectively sorted functionalproteins, lipids, and RNAs, mediating cell-to-cell communications and hence playing a role in thephysiology of the healthy and diseased organism. Challenges in the field include the identificationof mechanisms sustaining packaging of membrane-bound and soluble material to these vesicles andthe understanding of the underlying processes directing MVBs for degradation or fusion with theplasma membrane. The investigation into the formation and roles of exosomes in viral infection is inits early years. Although still controversial, exosomes can, in principle, incorporate any functionalfactor, provided they have an appropriate sorting signal, and thus are prone to viral exploitation.This review initially focuses on the composition and biogenesis of exosomes. It then explores theregulatory mechanisms underlying their biogenesis. Exosomes are part of the endocytic system,which is tightly regulated and able to respond to several stimuli that lead to alterations in thecomposition of its sub-compartments. We discuss the current knowledge of how these changesaffect exosomal release. We then summarize how different viruses exploit specific proteins ofendocytic sub-compartments and speculate that it could interfere with exosome function, althoughno direct link between viral usage of the endocytic system and exosome release has yet beenreported. Many recent reports have ascribed functions to exosomes released from cells infectedwith a variety of animal viruses, including viral spread, host immunity, and manipulation of themicroenvironment, which are discussed. Given the ever-growing roles and importance of exosomesin viral infections, understanding what regulates their composition and levels, and defining theirfunctions will ultimately provide additional insights into the virulence and persistence of infections. Full article
(This article belongs to the Special Issue Viruses and Exosomes)
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<p>The endocytic and secretory pathways. Cargo binds to the plasma membrane, is endocytosed by a plethora of processes and, independently of the entry route, is transported to early endosomes (EE). From this sub-compartment, cargo is sorted to one of three destinations: recycling, degradation, or secretion. These routes require maturation of the EE into recycling endosomes or multivesicular bodies (MVBs), which can either fuse with lysosomes (L) to generate endolysosomes (EL) or with the plasma membrane to release intraluminal vesicles to the milieu as exosomes. The membranes of the sub-compartments of the endocytic pathway have different compositions. Specific members of the Rab GTPase family, for example, are differentially enriched in each sub-compartment: Rab5 is enriched in EE; Rab7 in MVBs; Rab11, Rab25, Rab4, and Rab35 in the slow and rapid recycling routes; and Rab27a/b in MVBs. Rab9 is present in vesicles destined for retrograde transport to the trans-Golgi network (TGN). In uninfected cells, interfering with these Rabs affects exosome release. Many viruses use these Rabs in diverse steps of the viral life cycle, although whether this usage impacts in exosomal release has not been investigated. For example, at late stages of infection, viruses such as IAV, RSV, Sendai virus (SeV), and Andes virus (ANDV) were shown to hijack Rab11 vesicles to transport their progeny RNA to the cell surface. HIV, HSV1, and human cytomegalovirus (HCMV) were shown to require Rab27a/b vesicles for assembly. Human herpes 6 (HHV-6) virions were shown to be secreted upon fusion of MVB with the plasma membrane, together with exosomes.</p> Full article ">Figure 2
<p>Regulators of membrane identity. Membrane composition is important to maintain the integrity of endocytic process. Phosphoinositide (PI) kinases (and phosphatases) ensure the levels of specific PIPs in distinct membranes. These operate as docking platforms for guanine exchange and activator factors (GEFs and GAPs) able to recruit and turn on/off GTPases. GTPases involved in membrane integrity and vesicular biogenesis are mainly of two kinds: ADP ribosylation factors (ARFs) and ARF-like proteins (ARLs); and Rabs. ARFs and ARLs are involved in early steps of vesicular biogenesis such as recruiting coating proteins and cargo, membrane curvature, and neck formation. Rabs operate at later stages by recruiting effectors such as molecular motors, which are able to generate pulling forces and move released vesicles. Vesicle scission and release are mediated by highly specialized proteins that recognize, encircle, and cut the membrane neck, using GTP or ATP hydrolysis to drive the reaction.</p> Full article ">
715 KiB  
Review
Recent Progress in Therapeutic Treatments and Screening Strategies for the Prevention and Treatment of HPV-Associated Head and Neck Cancer
by Sonia N. Whang, Maria Filippova and Penelope Duerksen-Hughes
Viruses 2015, 7(9), 5040-5065; https://doi.org/10.3390/v7092860 - 17 Sep 2015
Cited by 36 | Viewed by 10167
Abstract
The rise in human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has elicited significant interest in the role of high-risk HPV in tumorigenesis. Because patients with HPV-positive HNSCC have better prognoses than do their HPV-negative counterparts, current therapeutic strategies for HPV [...] Read more.
The rise in human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has elicited significant interest in the role of high-risk HPV in tumorigenesis. Because patients with HPV-positive HNSCC have better prognoses than do their HPV-negative counterparts, current therapeutic strategies for HPV+ HNSCC are increasingly considered to be overly aggressive, highlighting a need for customized treatment guidelines for this cohort. Additional issues include the unmet need for a reliable screening strategy for HNSCC, as well as the ongoing assessment of the efficacy of prophylactic vaccines for the prevention of HPV infections in the head and neck regions. This review also outlines a number of emerging prospects for therapeutic vaccines, as well as for targeted, molecular-based therapies for HPV-associated head and neck cancers. Overall, the future for developing novel and effective therapeutic agents for HPV-associated head and neck tumors is promising; continued progress is critical in order to meet the challenges posed by the growing epidemic. Full article
(This article belongs to the Special Issue Tumour Viruses)
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<p>Involvement of small molecule inhibitors on cellular pathways affected by the E6 and E7 HPV oncoproteins.</p> Full article ">
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Article
Genetic Diversity and Selective Pressure in Hepatitis C Virus Genotypes 1–6: Significance for Direct-Acting Antiviral Treatment and Drug Resistance
by Lize Cuypers, Guangdi Li, Pieter Libin, Supinya Piampongsant, Anne-Mieke Vandamme and Kristof Theys
Viruses 2015, 7(9), 5018-5039; https://doi.org/10.3390/v7092857 - 16 Sep 2015
Cited by 58 | Viewed by 9975
Abstract
Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and [...] Read more.
Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1–6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity) and an additional 24% as pan-genotypic conserved (≥95%). Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%–0.46%) and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15), 33% (3/9), and 14% (2/14) of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%). NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may need to be genotype-specific or geographically tailored. Full article
(This article belongs to the Special Issue Bioinformatics and Computational Biology of Viruses)
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Graphical abstract

Graphical abstract
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<p>Phylogenetic tree of HCV full-genome sequences. A maximum-likelihood tree of HCV genotypes 1–7 was built using the GTR gamma model of substitution and the robustness of the tree was evaluated using 1000 bootstrap replicates. Bootstrap values above 70% are indicated at each main lineage, and the evolutionary distance scale bar indicates the number of nucleotide substitutions per site along each lineage.</p> Full article ">Figure 2
<p>Full-genome sliding window plot for within-genotype nucleotide diversity (%). A sliding window of 300 nucleotide positions with a step size of one nucleotide position was used. The six genotypes were plotted separately in color-coded solid lines (see figure legend). The genomic region of each protein is indicated at the bottom of the figure. Light-blue colored bars indicate genomic regions which are commonly sequenced.</p> Full article ">Figure 3
<p>Discretized frequencies of pan-genotypic consensus positions in the NS3, NS5A, and NS5B proteins. The distribution of positions that shared a consensus amino acid across genotypes 1–6, aligned against the reference sequence H77, is shown for HCV proteins NS3, NS5A, and NS5B. Genotype 1 is placed at the top and each square represents a single position. Positions that shared a consensus amino acid across all six genotypes were colored according to the frequency of the consensus amino acid in the respective genotype (for frequency x: category x &lt; 50% in red, 50% ≤ x &lt; 95% in orange, 95% ≤ x &lt; 99% in yellow and x ≥ 99% in green). Positions with different consensus amino acids are colored white and positions with no sequence data or a deletion are indicated in blue. It can be seen that the NS5B of HCV2 genomes are shorter compared to other genotypes.</p> Full article ">Figure 4
<p>dN/dS ratio at the full-length genome of all six HCV genotypes. Only positions characterized by a dN/dS ratio above 1 (and p-value &lt; 0.05) using SLAC, were defined as positively selected (<a href="#viruses-07-02857-s001" class="html-supplementary-material">Table S6</a>). A limited number of positions of the full-genome were identified as positively selected positions. X-axis: amino acid positions along the genome; Y-axis: dN/dS ratio; HCV proteins are shown at the bottom. For each HCV genotype, a line was drawn on the graph to indicate the dN/dS ratio equal to 1.</p> Full article ">
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Obituary
Jan van der Noordaa (1934–2015); A Virologist Pur Sang
by Ben Berkhout and Michael Bukrinsky
Viruses 2015, 7(9), 5016-5017; https://doi.org/10.3390/v7092859 - 15 Sep 2015
Viewed by 4574
Abstract
Our loyal friend and colleague, Jan van der Noordaa, passed away unexpectedly at the age of 80 on the evening of 17 June 2015. [...] Full article
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<p>Jan at“Il Palio” (Sienna, Italy, September 2013).</p> Full article ">
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Article
High Rate of Simian Immunodeficiency Virus (SIV) Infections in Wild Chimpanzees in Northeastern Gabon
by Vanina Boué, Sabrina Locatelli, Floriane Boucher, Ahidjo Ayouba, Christelle Butel, Amandine Esteban, Alain-Prince Okouga, Alphonse Ndoungouet, Peggy Motsch, Guillaume Le Flohic, Paul Ngari, Franck Prugnolle, Benjamin Ollomo, François Rouet and Florian Liégeois
Viruses 2015, 7(9), 4997-5015; https://doi.org/10.3390/v7092855 - 15 Sep 2015
Cited by 10 | Viewed by 6947
Abstract
The emergence of HIV-1 groups M, N, O, and P is the result of four independent cross-species transmissions between chimpanzees (cpz) and gorillas (gor) from central/south Cameroon and humans respectively. Although the first two SIVcpz were identified in wild-born captive chimpanzees in Gabon [...] Read more.
The emergence of HIV-1 groups M, N, O, and P is the result of four independent cross-species transmissions between chimpanzees (cpz) and gorillas (gor) from central/south Cameroon and humans respectively. Although the first two SIVcpz were identified in wild-born captive chimpanzees in Gabon in 1989, no study has been conducted so far in wild chimpanzees in Gabon. To document the SIVcpz infection rate, genetic diversity, and routes of virus transmission, we analyzed 1458 faecal samples collected in 16 different locations across the country, and we conducted follow-up missions in two of them. We found 380 SIV antibody positive samples in 6 different locations in the north and northeast. We determined the number of individuals collected by microsatellite analysis and obtained an adjusted SIV prevalence of 39.45%. We performed parental analysis to investigate viral spread between and within communities and found that SIVs were epidemiologically linked and were transmitted by both horizontal and vertical routes. We amplified pol and gp41 fragments and obtained 57 new SIVcpzPtt strains from three sites. All strains, but one, clustered together within a specific phylogeographic clade. Given that these SIV positive samples have been collected nearby villages and that humans continue to encroach in ape’s territories, the emergence of a new HIV in this area needs to be considered. Full article
(This article belongs to the Section Animal Viruses)
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<p>Geographical distribution of chimpanzee faecal samples collection sites (white and yellow circles) from Gabon Locations of faecal samples collection: LO = Louango, WA = Waka, MK = Makande, MI = Mikongo, LP = Lopé National Park, LN = Lopé SEGC site, MC = Monts de Cristal, OY = Oyem, IV = Ivindo, DJ = Djidji, LA = Langoué, TS = Tsouba, OD = Odjala, Ma = Makatamangoye, IY = Iyoko milieu, ML = Malouma. Yellow circles represent sites where SIVcpz positive samples were identified.</p> Full article ">Figure 2
<p>SIV antibody positive profiles. Example of HIV-1/HIV-2 cross-reactive antibodies profile in chimpanzee faecal samples using a line immunoassay (INNO-LIA HIV Confirmation, Innogenetics, Ghent, Belgium). Varying patterns of reactivity to HIV peptides and proteins (gp41, p24, p31 and p17) are shown. Plasma samples from HIV-1/HIV-2-negative and -positive persons are shown as controls on the left. The 3+, 1+ and +/− bands at the top of all test strips control for sample addition (presence of plasma immunoglobulin) and test performance (binding of secondary antibody).</p> Full article ">Figure 3
<p>SIVcpz antibody positive samples from north-east Gabon. A/ Yellow circles with red perimeter represent sampling sites where SIVcpz have been PCR amplified. B/ details of SIVcpz positive sampling sites: colored circles correspond to the color codes used to represent the different SIVcpz identified in <a href="#viruses-07-02855-f004" class="html-fig">Figure 4</a> and <a href="#viruses-07-02855-f005" class="html-fig">Figure 5</a>: Blue = MA, Green = IY and Orange = ML. Triangles correspond to SIVcpz antibody positive faecal samples collected in OD, IY and ML. Dots correspond to SIVcpz antibody positive faecal samples collected in MA. Prevalence based on serological tests is showed for each site. The trail linking Odjala to Malouma is represented by a red line. The green area corresponds to the different national parks. The site of DJ is not represented.</p> Full article ">Figure 4
<p>Phylogenetic analysis of partial <span class="html-italic">pol</span> (A) and <span class="html-italic">gp41</span> (B) of the newly identified SIVcpz sequences from Gabon. New partial <span class="html-italic">pol</span> (150bp) and <span class="html-italic">gp41</span> (170bp) SIVcpz-Gab sequences were compared to previously identified SIVcpz<span class="html-italic">Ptt</span> and SIVcpz<span class="html-italic">Pts</span> as well as HIV-1 goups M, N, O and P. Phylogenies were inferred using Neighbor-Joining method implemented in Mega 5 with the Kimura 2 Parameters model of evolution. Asterisks at nodes represent bootstraps values ≥70% (100 replicates). Scale bars indicate the number of base substitutions per site. New SIVcpz strains are colour-coded in accordance with figure 2B (MA in blue, ML in orange and IY in green). Strains amplified from genotyped animals are named using their ID number (IDXXX), whereas strains amplified from non-genotyped samples are named using the sample field number (GabXXXX). SIVcpz<span class="html-italic">Ptt</span>-Gab-1, -2 and -4 are shown in bold. HIV-1 groups M, N, O and P are shown in red.</p> Full article ">Figure 5
<p>Phylogenetic analysis of partial <span class="html-italic">pol</span> (A), <span class="html-italic">env</span> (B) and <span class="html-italic">gp41-nef</span> (C) of the newly identified SIVcpz sequences from Gabon. New partial <span class="html-italic">pol</span> (365bp), <span class="html-italic">env (325bp)</span> and <span class="html-italic">gp41-nef</span> (916bp) SIVcpz-GAB sequences were compared to previously identified SIVcpz<span class="html-italic">Ptt</span> and SIVcpz<span class="html-italic">Pts</span> as well as HIV-1 goups M, N, O and P. Phylogenies were inferred using Maximum Likelihood methods implemented in PhyML under the GTR+Γ<sub>4</sub>+I model of evolution. Asterisks at nodes represent bootstrap values ≥70% (1000 replicates). Scale bars indicate the number of base substitutions per site. New SIVcpz strains are colour-coded in accordance with figure 2B (MA in blue and ML in orange). Strains amplified from genotyped animals are named using their ID number (IDXXX), whereas strains amplified from non-genotyped samples are named using the sample field number (GabXXXX). SIVcpz<span class="html-italic">Ptt</span>-Gab-1, -2 and -4 are shown in bold. HIV-1 groups M, N, O and P are shown in red.</p> Full article ">
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Article
Alterations of Nuclear Architecture and Epigenetic Signatures during African Swine Fever Virus Infection
by Margarida Simões, José Rino, Inês Pinheiro, Carlos Martins and Fernando Ferreira
Viruses 2015, 7(9), 4978-4996; https://doi.org/10.3390/v7092858 - 15 Sep 2015
Cited by 28 | Viewed by 8022
Abstract
Viral interactions with host nucleus have been thoroughly studied, clarifying molecular mechanisms and providing new antiviral targets. Considering that African swine fever virus (ASFV) intranuclear phase of infection is poorly understood, viral interplay with subnuclear domains and chromatin architecture were addressed. Nuclear speckles, [...] Read more.
Viral interactions with host nucleus have been thoroughly studied, clarifying molecular mechanisms and providing new antiviral targets. Considering that African swine fever virus (ASFV) intranuclear phase of infection is poorly understood, viral interplay with subnuclear domains and chromatin architecture were addressed. Nuclear speckles, Cajal bodies, and promyelocytic leukaemia nuclear bodies (PML-NBs) were evaluated by immunofluorescence microscopy and Western blot. Further, efficient PML protein knockdown by shRNA lentiviral transduction was used to determine PML-NBs relevance during infection. Nuclear distribution of different histone H3 methylation marks at lysine’s 9, 27 and 36, heterochromatin protein 1 isoforms (HP1α, HPβ and HPγ) and several histone deacetylases (HDACs) were also evaluated to assess chromatin status of the host. Our results reveal morphological disruption of all studied subnuclear domains and severe reduction of viral progeny in PML-knockdown cells. ASFV promotes H3K9me3 and HP1β foci formation from early infection, followed by HP1α and HDAC2 nuclear enrichment, suggesting heterochromatinization of host genome. Finally, closeness between DNA damage response factors, disrupted PML-NBs, and virus-induced heterochromatic regions were identified. In sum, our results demonstrate that ASFV orchestrates spatio-temporal nuclear rearrangements, changing subnuclear domains, relocating Ataxia Telangiectasia Mutated Rad-3 related (ATR)-related factors and promoting heterochromatinization, probably controlling transcription, repressing host gene expression, and favouring viral replication. Full article
(This article belongs to the Section Animal Viruses)
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<p>(<b>A</b>) ASFV induces the reorganization of subnuclear domains. Vero cells were infected with ASFV Ba71V isolate (MOI of 5), fixed at 6 hpi, permeabilized and immunostained; cell nuclei stained with DAPI (blue). (<b>a</b>–<b>d</b>) ASFV-infected cells (green) reveal globular and enlarged accumulations of SC-35 (red), while in non-infected cells (<b>e</b>–<b>h</b>), nuclear speckles (SC-35) show a pan-nuclear staining. (<b>i</b>–<b>l</b>) In ASFV-infected Vero cells (green), Cajal bodies (coilin, red) display “comma-shaped” morphology and group together, contrasting with the few pin-point bright foci of non-infected cells (<b>m</b>–<b>p</b>). (<b>q</b>–<b>t</b>) PML-NBs (PML, red) of infected cells (green) reveal fewer and enlarged domains, when compared to non-infected cells which show an increased number of smaller dots (<b>u</b>–<b>z</b>). Scale bar, 10 μm. Representative images of at least three independent experiments are shown; (<b>B</b>) Protein levels of SC-35, coilin and PML remain constant during ASFV infection. Vero cells infected with ASFV Ba71V isolate were lysed at 6 and 12 hpi, and compared to mock-infected cells, using immunoblotting analysis. α-Tubulin was used as loading control. Molecular weights (kDa) of evaluated proteins are indicated on the left of immunoblot images.</p> Full article ">Figure 2
<p>PML-NBs and DDR factors juxtapose during ASFV infection. (<b>A</b>) Immunofluorescence analysis of PML-NBs (cyan) and two DNA damage response factors (red) was performed in ASFV-infected cells (8 hpi, green) and in non-infected cells. Cell nuclei were counterstained with DAPI (blue). (<b>a</b>–<b>d</b>) In ASFV-infected cells, enlarged PML-NBs (cyan) are juxtaposed to phosphorylated p53 form (p-p53, red), while in non-infected cells these subnuclear domains do not associate to activated p53 loci (<b>e</b>–<b>h</b>). Additionally, enlarged PML-NBs neighbour pATR accumulations (<b>i</b>–<b>l</b>), as non-infected cells display smaller PML-NBs and pATR faint staining dispersed throughout the nucleus (<b>m</b>–<b>p</b>). Scale bar, 10 μm. Representative microscopy images of at least three independent experiments are shown; (<b>B</b>) and (<b>C</b>) Relative distance between PML-NBs and p-p53/pATR foci was evaluated by radial intensity profile analysis in ASFV-infected cells (solid lines) and non-infected cells (dashed lines). Normalized fluorescence intensity curves from the centre of PML-NBs (blue lines) to p-p53 accumulations (B) or to pATR foci (C) (represented by red lines) are plotted. Error bars represent standard errors (±SE). Radial profile analysis shows the close proximity between the subnuclear domains and the higher intensity DDR-factor accumulation regions only in ASFV-infected cells, as blue and red solid lines cross at a point of the studied radius, contrasting to the absence of intersection between PML-NBs (blue dashed lines) and p-p53/pATR fluorescence intensities (red dashed lines) in non-infected cells.</p> Full article ">Figure 3
<p>PML has a proviral role in ASFV infection. (<b>A</b>) Whole cell extracts were collected from Vero-shRNA-PML and Vero-shRNA-GAPDH cells, non-infected and infected with ASFV Ba71V isolate (MOI of 1) at 6, 12 and 18 hpi. In Vero-shRNA-PML cells, expression levels of a ≈22 kDa viral protein remain residual throughout infection, whereas in Vero-shRNA-GAPDH cells (control) this viral protein showed increasing levels. In addition, other structural viral proteins (≈32 kDa and ≈72 kDa) in ASFV-infected PML knockdown cells did not present the expression levels detected in infected shGAPDH cells, even at late times of infection. Overall, viral protein synthesis is diminished in Vero-shRNA-PML knockdown cells. As expected, mock-infected cells (0 hpi), showed no viral protein expression. α-Tubulin was used as loading control. Molecular weights (kDa) of evaluated proteins are indicated on the left of immunoblot images; (<b>B</b>) PML knockdown cells display lower intensity viral proteins staining and aberrant ASFV factories. (<b>a</b>–<b>d</b>) Vero-shRNA-PML cells (GFP expressing, green) were infected with ASFV (MOI of 1) and analysed at 12 hpi. PML (cyan) and ASFV-infected cells (red) were further detected by immunofluorescence. PML-NBs could not be visualized in PML knockdown cells, which show viral cytoplasmic factories with atypical morphology (horseshoe-shaped). (<b>e</b>–<b>h</b>) In contrast, Vero-shRNA-GAPDH cells (green) display enlarged PML-NBs and typical round-shaped viral factories (red). Scale bar, 10 µm. Representative images of at least three independent experiments are shown; (<b>C</b>) ASFV progeny is reduced in Vero-shRNA-PML kd cells. A drastic reduction in virus yields was observed in ASFV-infected PML knockdown cells (light grey columns) in comparison to the viral progeny production obtained from infected Vero-shRNA-GAPDH cells (dark grey columns). Each column represents the average of results obtained from three independent experiments, and the error bars represent the standard error (SE) values. Log decay of virus titer was considered as statistically significant (<span class="html-italic">p</span> value &lt; 0.05).</p> Full article ">Figure 4
<p>ASFV modifies the host chromatin state. Immunofluorescence analysis was performed on non-infected and ASFV-infected Vero cells (8 hpi) using specific antibodies recognizing heterochromatin marks—H3K9me3, HP1β and HDAC2 (red) and viral proteins (green). Cell nuclei were counterstained with DAPI (blue). (<b>a</b>–<b>d</b>) Histone H3 trimethylated at lysine 9 (H3K9me3, red) show large accumulations throughout the nucleoplasm upon ASFV infection (green), not observed in uninfected cells (<b>e</b>–<b>h</b>). ASFV-infected cells (green) also present HP1β (red) nucleoplasmic accumulations (<b>i</b>–<b>l</b>), not detected in non-infected cells (<b>m</b>–<b>p</b>). HDAC2 (red) is the only member of HDACs family that displays a more intense nuclear labelling in ASFV-infected cells (green), and additional recruitment to viral cytoplasmic factories (<b>q</b>–<b>t</b>), when compared to non-infected cells (<b>u</b>–<b>z</b>). Scale bar, 10 μm. Representative microscopy images of at least three independent experiments are shown.</p> Full article ">Figure 5
<p>ASFV leads to juxtaposition of host heterochromatic regions, disrupted subnuclear domains and pATR foci. (<b>A</b>) Heterochromatin marker HP1β is labelled in red, while subnuclear domains (PML-NBs, nuclear speckles or Cajal bodies) and the activated ATR kinase are labelled in cyan. Cell nuclei were counterstained with DAPI (blue). (<b>a</b>–<b>d</b>) Only ASFV-infected cells (green) display the speckled pattern of enlarged SC-35 accumulations (cyan) juxtaposed to the heterochromatic regions (HP1 β, red), as non-infected cells do not reveal the close proximity pattern (<b>a’</b>–<b>d’</b>). In contrast to non-infected cells (<b>e’</b>–<b>h’</b>), the ASFV-induced bulky heterochromatic territories (HP1β, red) are always present within close vicinity to disrupted Cajal bodies (cyan) (<b>e</b>–<b>h</b>). Enlarged heterochromatic regions (HP1β, red) closely neighbour reorganized PML-NBs (cyan) in infected cells (green) (<b>i</b>–<b>l)</b>, rearrangements not observable in non-infected cells (<b>i'</b>–<b>l’</b>). HP1β deposits (red) juxtapose to pATR foci (cyan), in ASFV-infected cells (green) (<b>m</b>–<b>p</b>), different chromatin/pATR appearances and distributions in non-infected cells (m’–p’). Scale bar, 10 μm. Representative microscopy images of at least three independent experiments are shown; (<b>B</b>–<b>E</b>) Relative distance between the studied subnuclear domains/pATR foci (cyan) and heterochromatic regions (red) was evaluated by radial intensity profile analysis in ASFV-infected cells (solid lines) and non-infected cells (dashed lines). Normalized fluorescence curves were obtained from 50 nuclei. Experiments were performed in triplicate and error bars represent standard errors (±SE). Radial profile analysis shows the close proximity, in ASFV-infected cells, between each subnuclear domain/pATR foci (blue solid line) and HP1β accumulations (red solid line), crossing at a point within the given radius (1 µm). Dashed lines representing fluorescence intensities of subnuclear domains/pATR (blue) and heterochromatic regions (red) never intersect, revealing a greater distance between these nuclear domains/factors in non-infected cells.</p> Full article ">Figure 5 Cont.
<p>ASFV leads to juxtaposition of host heterochromatic regions, disrupted subnuclear domains and pATR foci. (<b>A</b>) Heterochromatin marker HP1β is labelled in red, while subnuclear domains (PML-NBs, nuclear speckles or Cajal bodies) and the activated ATR kinase are labelled in cyan. Cell nuclei were counterstained with DAPI (blue). (<b>a</b>–<b>d</b>) Only ASFV-infected cells (green) display the speckled pattern of enlarged SC-35 accumulations (cyan) juxtaposed to the heterochromatic regions (HP1 β, red), as non-infected cells do not reveal the close proximity pattern (<b>a’</b>–<b>d’</b>). In contrast to non-infected cells (<b>e’</b>–<b>h’</b>), the ASFV-induced bulky heterochromatic territories (HP1β, red) are always present within close vicinity to disrupted Cajal bodies (cyan) (<b>e</b>–<b>h</b>). Enlarged heterochromatic regions (HP1β, red) closely neighbour reorganized PML-NBs (cyan) in infected cells (green) (<b>i</b>–<b>l)</b>, rearrangements not observable in non-infected cells (<b>i'</b>–<b>l’</b>). HP1β deposits (red) juxtapose to pATR foci (cyan), in ASFV-infected cells (green) (<b>m</b>–<b>p</b>), different chromatin/pATR appearances and distributions in non-infected cells (m’–p’). Scale bar, 10 μm. Representative microscopy images of at least three independent experiments are shown; (<b>B</b>–<b>E</b>) Relative distance between the studied subnuclear domains/pATR foci (cyan) and heterochromatic regions (red) was evaluated by radial intensity profile analysis in ASFV-infected cells (solid lines) and non-infected cells (dashed lines). Normalized fluorescence curves were obtained from 50 nuclei. Experiments were performed in triplicate and error bars represent standard errors (±SE). Radial profile analysis shows the close proximity, in ASFV-infected cells, between each subnuclear domain/pATR foci (blue solid line) and HP1β accumulations (red solid line), crossing at a point within the given radius (1 µm). Dashed lines representing fluorescence intensities of subnuclear domains/pATR (blue) and heterochromatic regions (red) never intersect, revealing a greater distance between these nuclear domains/factors in non-infected cells.</p> Full article ">Figure 6
<p>Proposed working model for ASFV-host interactions. ASFV-infected cells show a fully reorganized nuclear architecture. ASFV genomes most probably recognized as DNA damage sites, after migrating into host cell nucleus, activate DNA damage response factors (p-p53 and pATR), that juxtapose to enlarged PML-NBs (yellow circles). The activated p53 (p-p53, purple circles) and ATR (pATR, red shapes) also accumulate nearby heterochromatic regions (blue forms). In addition, the viral infection promotes nuclear speckles enlargement (pink circles) and Cajal bodies remodelling (green “comma-shaped” forms). All subnuclear domains display close vicinity to viral-induced heterochromatic regions enriched by H3K9me3, H3K27me3, HP1α/β isoforms and HDAC2.</p> Full article ">
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Review
Anti-HBV Drugs: Progress, Unmet Needs, and New Hope
by Lei Kang, Jiaqian Pan, Jiaofen Wu, Jiali Hu, Qian Sun and Jing Tang
Viruses 2015, 7(9), 4960-4977; https://doi.org/10.3390/v7092854 - 15 Sep 2015
Cited by 41 | Viewed by 8346
Abstract
Approximately 240 million people worldwide are chronically infected with hepatitis B virus (HBV), which represents a significant challenge to public health. The current goal in treating chronic HBV infection is to block progression of HBV-related liver injury and inflammation to end-stage liver diseases, [...] Read more.
Approximately 240 million people worldwide are chronically infected with hepatitis B virus (HBV), which represents a significant challenge to public health. The current goal in treating chronic HBV infection is to block progression of HBV-related liver injury and inflammation to end-stage liver diseases, including cirrhosis and hepatocellular carcinoma, because we are unable to eliminate chronic HBV infection. Available therapies for chronic HBV infection mainly include nucleos/tide analogues (NAs), non-NAs, and immunomodulatory agents. However, none of them is able to clear chronic HBV infection. Thus, a new generation of anti-HBV drugs is urgently needed. Progress has been made in the development and testing of new therapeutics against chronic HBV infection. This review aims to summarize the state of the art in new HBV drug research and development and to forecast research and development trends and directions in the near future. Full article
(This article belongs to the Special Issue Viral Replication Complexes: Structures, Functions, Applications and Inhibitors)
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Article
Determinants of Disease Phenotype Differences Caused by Closely-Related Isolates of Begomovirus Betasatellites Inoculated with the Same Species of Helper Virus
by Jie Zhang, Mingqing Dang, Qingqing Huang and Yajuan Qian
Viruses 2015, 7(9), 4945-4959; https://doi.org/10.3390/v7092853 - 14 Sep 2015
Cited by 4 | Viewed by 5269
Abstract
Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus associated with different betasatellites. In this study, we investigate two different isolates of Tomato yellow leaf curl China betasatellite (TYLCCNB) to determine what features of the viral genome are required for induction [...] Read more.
Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus associated with different betasatellites. In this study, we investigate two different isolates of Tomato yellow leaf curl China betasatellite (TYLCCNB) to determine what features of the viral genome are required for induction of characteristic phenotypic differences between closely-related betasatellite. When co-agroinoculated with TYLCCNV into Nicotiana spp. and tomato plants, TYLCCNB-Y25 induced only leaf curling on all hosts, while TYLCCNB-Y10 also induced enations, vein yellowing, and shoot distortions. Further assays showed that βC1 of TYLCCNB-Y25 differs from that of TYLCCNB-Y10 in symptom induction and transcriptional modulating. Hybrid satellites were constructed in which the βC1 gene or 200 nt partial promoter-like fragment upstream of the βC1 were exchanged. Infectivity assays showed that a TYLCCNB-Y25 hybrid with the intact TYLCCNB-Y10 βC1 gene was able to induce vein yellowing, shoot distortions, and a reduced size and number of enations. A TYLCCNB-Y10 hybrid with the intact TYLCCNB-Y25 βC1 gene produced only leaf curling. In contrast, the TYLCCNB-Y25 and TYLCCNB-Y10 hybrids with swapped partial promoter-like regions had little effect on the phenotypes induced by wild-type betasatellites. Further experiments showed that the TYLCCNB-Y25 hybrid carrying the C-terminal region of TYLCCNB-Y10 βC1 induced TYLCCNB-Y10-like symptoms. These findings indicate that the βC1 protein is the major symptom determinant and that the C-terminal region of βC1 plays an important role in symptom induction. Full article
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<p>Symptoms induced by co-inoculation with TYLCCNV with TYLCCNB-Y10 or TYLCCNB-Y25 satellites in <span class="html-italic">N. benthamiana</span>, <span class="html-italic">N. glutinosa</span>, <span class="html-italic">N. tabacum</span> cv. Samsun and <span class="html-italic">S. lycopersicum</span>. (<b>A</b>) Whole plants. Photographs of plants were taken at 30 days post inoculation (dpi); (<b>B</b>) abaxial surfaces of leaves. Arrowheads indicate enations; (<b>C</b>) Southern blot analysis for viral DNA in <span class="html-italic">N. benthamiana</span>, <span class="html-italic">N. glutinosa</span>, <span class="html-italic">N. tabacum</span> cv. Samsun, and <span class="html-italic">S. lycopersicum</span> plants agroinoculated with TYLCCNV along with TYLCCNB-Y10 or TYLCCNB-Y25. The blots were probed either for TYLCCNV (top) or for betasatellite (bottom). The lower panel represents an ethidium bromide-stained gel of DNA samples as a loading control. Y10β, TYLCCNB-Y10; Y25β, TYLCCNB-Y25.</p> Full article ">Figure 2
<p>Phenotypes and Northern blot analysis of transgenic plants containing the TYLCCNB <span class="html-italic">βC1</span> gene. Phenotypes of transgenic tobacco transformed with 35S-Y25βC1, symptomatic plants (<b>A</b> and <b>B</b>); symptomless (<b>C</b> and <b>D</b>); 35S-Y10βC1 (<b>E</b> and <b>F</b>); (<b>G</b>) Northern blot analysis. Lanes 1–4, 35S-Y10βC1; lanes 5–7, 35S-Y25βC1 (symptomatic plants); lanes 8,9, 35S-Y25βC1 (symptomless plants); lane 10, pCHF3 empty vector. The ethidium bromide-stained gel shown below the blot indicates equal loading of total RNA.</p> Full article ">Figure 3
<p>The nucleotide sequence encompassing the entire non-coding region (982 nt) upstream of the TYLCCNB-Y25 <span class="html-italic">βC1</span> open reading frame. The translation start site A is labeled +1. The putative motifs are shown in frame or marked by underline.</p> Full article ">Figure 4
<p>Identification of promoter activity in <span class="html-italic">N</span>. <span class="html-italic">benthamiana</span> leaves after transiently expressing various TYLCCNB-Y25 <span class="html-italic">β</span><span class="html-italic">C1</span>-derived promoters. (<b>A</b>) Schematic representation of the TYLCCNB-Y25 genome and various <span class="html-italic">β</span><span class="html-italic">C1</span> promoters fused to a promoter-less pINT121 or pCHF3:GFP vector; (<b>B</b>) fluorometric activity analysis after transient expression various promoter constructs. The mean GUS or GFP activity from the CaMV 35S promoter of pINT121 or pCHF3:GFP was considered as 100% and used to standardize the activities for all of the other constructs, respectively. Columns represent the mean value, with standard error of the mean. The significant difference between treatments (** <span class="html-italic">p</span> ≤ 0.01) was shown; (<b>C</b>) confocal microscopy showed GFP fluorescence after transient expression various promoter constructs.</p> Full article ">Figure 5
<p>Symptoms induced by co-inoculation with TYLCCNV and either wild-type or hybrid satellites of TYLCCNB-Y10 and TYLCCNB-Y25 in <span class="html-italic">N. benthamiana</span>. (<b>A</b>) Whole plants. Photographs of plants were taken at 30 dpi; (<b>B</b>) abaxial surfaces of leaves. Arrowheads indicate enations; (<b>C</b>) Southern blot analysis for viral DNA in <span class="html-italic">N. benthamiana</span> plants. The blots were probed either for TYLCCNV (top) or for betasatellite (bottom). The lower panel represents an ethidium bromide-stained gel of DNA samples as a loading control.</p> Full article ">Figure 6
<p>Alignment of <span class="html-italic">βC1</span> nucleotide sequence (<b>A</b>) and predicted βC1 amino acid sequence (<b>B</b>) from TYLCCNB-Y10 and TYLCCNB-Y25. Arrowheads indicate the split sites.</p> Full article ">Figure 7
<p>Symptoms induced by co-inoculation with TYLCCNV with hybrid betasatellites of TYLCCNB-Y10 and TYLCCNB-Y25 in <span class="html-italic">N. benthamiana</span>. (<b>A</b>) Whole plants. Photographs of plants were taken at 30 dpi; (<b>B</b>) abaxial leaf surfaces. Arrowheads indicate enations; (<b>C</b>) Southern blot analysis for viral DNA in <span class="html-italic">N. benthamiana</span> plants. The blots were probed either for TYLCCNV (top) or for betasatellite (bottom). The lower panel represents an ethidium bromide-stained gel of DNA samples as a loading control.</p> Full article ">Figure 8
<p>Schematic representation showing the organization of hybrid betasatellites. Betasatellite organization is shown as linear DNA in the complementary sense. SCR, satellite conserved region; A-rich, adenine-rich; βC1, <span class="html-italic">βC1</span> gene; TYLCCNB-Y10, tomato yellow leaf curl China betasatellite-Y10; TYLCCNB-Y25, tomato yellow leaf curl China betasatellite-Y25.</p> Full article ">
646 KiB  
Review
Clinical Implications of Antiviral Resistance in Influenza
by Timothy C. M. Li, Martin C. W. Chan and Nelson Lee
Viruses 2015, 7(9), 4929-4944; https://doi.org/10.3390/v7092850 - 14 Sep 2015
Cited by 160 | Viewed by 27429
Abstract
Influenza is a major cause of severe respiratory infections leading to excessive hospitalizations and deaths globally; annual epidemics, pandemics, and sporadic/endemic avian virus infections occur as a result of rapid, continuous evolution of influenza viruses. Emergence of antiviral resistance is of great clinical [...] Read more.
Influenza is a major cause of severe respiratory infections leading to excessive hospitalizations and deaths globally; annual epidemics, pandemics, and sporadic/endemic avian virus infections occur as a result of rapid, continuous evolution of influenza viruses. Emergence of antiviral resistance is of great clinical and public health concern. Currently available antiviral treatments include four neuraminidase inhibitors (oseltamivir, zanamivir, peramivir, laninamivir), M2-inibitors (amantadine, rimantadine), and a polymerase inhibitor (favipiravir). In this review, we focus on resistance issues related to the use of neuraminidase inhibitors (NAIs). Data on primary resistance, as well as secondary resistance related to NAI exposure will be presented. Their clinical implications, detection, and novel therapeutic options undergoing clinical trials are discussed. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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<p>Molecular targets and potential antiviral treatments against influenza virus infection. The above diagram shows the life cycle of influenza virus and the proposed action of each class of antiviral. After attachment to the host cell receptor containing sialic acid, the virus particle undergoes the processes of fusion, endocytosis, and uncoating, and subsequently replication by the RNA polymerase. Surface protein-coated envelope then forms around the genome to produce a complete virion, which can then be released to infect other cells. DAS 181, a sialidase fusion protein, acts on the first step of virus invasion by cleaving the sialic acid linkages on human epithelial cells. Adamantanes are M2 channel blockers which inhibit proton entry through the channel into the virion, thus preventing its disintegration. Favipiravir is a pyrazinecarboxamide derivative which inhibits the viral RNA-dependent RNA polymerase. Ribavirin’s antiviral actions are multiple, though it mainly interferes with RNA synthesis. Nitazoxanide may block haemagglutinin maturation (and act as an interferon-inducer). Neuraminidase inhibitors, by attaching to the viral neuraminidase, block the release of virus from host cells, thus halting the progression of infection. A combination of agents from different drug classes may produce synergistic effects (see text).</p> Full article ">
1262 KiB  
Review
Insect-Specific Virus Discovery: Significance for the Arbovirus Community
by Bethany G. Bolling, Scott C. Weaver, Robert B. Tesh and Nikos Vasilakis
Viruses 2015, 7(9), 4911-4928; https://doi.org/10.3390/v7092851 - 10 Sep 2015
Cited by 202 | Viewed by 15667
Abstract
Arthropod-borne viruses (arboviruses), especially those transmitted by mosquitoes, are a significant cause of morbidity and mortality in humans and animals worldwide. Recent discoveries indicate that mosquitoes are naturally infected with a wide range of other viruses, many within taxa occupied by arboviruses that [...] Read more.
Arthropod-borne viruses (arboviruses), especially those transmitted by mosquitoes, are a significant cause of morbidity and mortality in humans and animals worldwide. Recent discoveries indicate that mosquitoes are naturally infected with a wide range of other viruses, many within taxa occupied by arboviruses that are considered insect-specific. Over the past ten years there has been a dramatic increase in the literature describing novel insect-specific virus detection in mosquitoes, which has provided new insights about viral diversity and evolution, including that of arboviruses. It has also raised questions about what effects the mosquito virome has on arbovirus transmission. Additionally, the discovery of these new viruses has generated interest in their potential use as biological control agents as well as novel vaccine platforms. The arbovirus community will benefit from the growing database of knowledge concerning these newly described viral endosymbionts, as their impacts will likely be far reaching. Full article
(This article belongs to the Special Issue Impact of the Insect Microbiome on Arbovirus Transmission)
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<p>Timeline demonstrating dramatic increase in insect-specific virus discovery.</p> Full article ">Figure 2
<p>Flaviviruses: Maximum-likelihood analysis of select members of the genus <span class="html-italic">flavivirus</span>. Scale bars indicate amino acid substitutions/site. Branch labels indicate virus abbreviation. Additional virus isolate information is contained in a <a href="#viruses-07-02851-s001" class="html-supplementary-material">supplemental file</a>.</p> Full article ">Figure 3
<p>Rhabdovirus: Maximum-likelihood (ML) analysis of rhabdovirus L protein sequences. Horizontal branch lengths are drawn to a scale of amino acid substitutions/site, and all bootstrap support values ≥85% are indicated by an asterisk. Cytorhabdovirus, novirhabdovirus and nucleorhabdovirus outgroup sequences were excluded from the tree as they were too divergent to establish a reliable rooting. The tree is therefore rooted arbitrarily on one of two basal clades (genera Almendravirus and Bahiavirus) that comprise viruses isolated from mosquitoes. Branch labels indicate virus abbreviation. Additional virus isolate information is contained in a <a href="#viruses-07-02851-s001" class="html-supplementary-material">supplemental file</a>. (Adapted from [<a href="#B73-viruses-07-02851" class="html-bibr">73</a>]).</p> Full article ">Figure 4
<p>Mesoniviruses: Maximum-likelihood analysis of conserved protein domains of ORF1ab (3CLpro, RdRp, HEL1). Scale bars indicate amino acid substitutions/site. Branch labels indicate virus abbreviation/strain. Additional virus isolate information is contained in a <a href="#viruses-07-02851-s001" class="html-supplementary-material">supplemental file</a>. (Adapted from [<a href="#B44-viruses-07-02851" class="html-bibr">44</a>]).</p> Full article ">Figure 5
<p>Negeviruses: Maximum likelihood analysis of select negeviruses. The region of the genome corresponds to the nt 4316–7309 (Negev E0239), which corresponds to the RNA-dependent RNA-polymerase of the genome. Branch labels indicate virus abbreviation/strain. Additional virus isolate information is contained in a <a href="#viruses-07-02851-s001" class="html-supplementary-material">supplemental file</a>.</p> Full article ">Figure 6
<p>Togaviruses: Maximum likelihood analysis of select togaviruses. Phylogenetic tree based on nucleotide sequences of the alphavirus structural ORF. Branch labels indicate virus abbreviation/strain. Additional virus isolate information is contained in a <a href="#viruses-07-02851-s001" class="html-supplementary-material">supplemental file</a>.</p> Full article ">Figure 7
<p>Mosquito vector competence for arboviruses can be affected by external and internal factors.</p> Full article ">
3710 KiB  
Communication
Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection
by Chih-Yeu Fang, Siang-Jyun Chen, Huey-Nan Wu, Yueh-Hsin Ping, Ching-Yen Lin, David Shiuan, Chi-Long Chen, Ying-Ray Lee and Kao-Jean Huang
Viruses 2015, 7(9), 4894-4910; https://doi.org/10.3390/v7092852 - 10 Sep 2015
Cited by 63 | Viewed by 8513
Abstract
Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV) infection. Honokiol, a lignan [...] Read more.
Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV) infection. Honokiol, a lignan biphenol derived from the Magnolia tree, is commonly used in Eastern medicine. Here we report that honokiol has profound antiviral activity against serotype 2 DENV (DENV-2). In addition to inhibiting the intracellular DENV-2 replicon, honokiol was shown to suppress the replication of DENV-2 in baby hamster kidney (BHK) and human hepatocarcinoma Huh7 cells. At the maximum non-toxic dose of honokiol treatment, the production of infectious DENV particles was reduced >90% in BHK and Huh7 cells. The underlying mechanisms revealed that the expression of DENV-2 nonstructural protein NS1/NS3 and its replicating intermediate, double-strand RNA, was dramatically reduced by honokiol treatment. Honokiol has no effect on the expression of DENV putative receptors, but may interfere with the endocytosis of DENV-2 by abrogating the co-localization of DENV envelope glycoprotein and the early endosomes. These results indicate that honokiol inhibits the replication, viral gene expression, and endocytotic process of DENV-2, making it a promising agent for chemotherapy of DENV infection. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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<p>Honokiol inhibits DENV (dengue virus) replicon. (<b>A</b>) The chemical structure of honokiol; (<b>B</b>) BHK (baby hamster kidney) cells were treated with various concentrations of honokiol (1–20 μM) for 24 h, and the viability of BHK cells was measured by MTT assay; (<b>C</b>) The luciferase activity was measured in cell lysates after treatment with honokiol (1–20 μM) for 24 h. Data indicate the average value of triplicates (mean ± SD). ** <span class="html-italic">p</span> = 0.006; *** <span class="html-italic">p</span> &lt; 0.001, as compared with the vehicle control.</p> Full article ">Figure 2
<p>Honokiol decreases dengue virus production. (<b>A</b>) The cytotoxicity of honokiol on BHK and Huh7 cells was measured by MTT assay. Various concentrations of honokiol were applied to cells for 48 h; (<b>B</b>,<b>C</b>) Infectious DENV-2 particles released from DENV-infected, mock/honokiol-treated (<b>B</b>) BHK and (<b>C</b>) Huh7 cells were determined by fluorescence focus assay. Quantification of the virus number was calculated by (fluorescence focus units) × (dilution factor) × (total supernatant volume) and plotted as a bar chart. Data indicate the average value of triplicates (mean ± SD). * <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001, as compared with the control.</p> Full article ">Figure 3
<p>Honokiol inhibits dengue virus translation and replication in cells. (<b>A</b>) Immunofluorescence assay of honokiol-treated, DENV-infected BHK cells with MOI = 0.1. The viral NS1, NS3, and dsRNA antigens were detected after 48 h of honokiol treatment; (<b>B</b>) Immunofluorescence assay of honokiol-treated, DENV-infected Huh7 cells with MOI = 10. The viral NS1, NS3, and dsRNA antigens were detected after 48 h of honokiol treatment; (<b>C</b>, <b>D</b>) The percentage of viral NS1, NS3, and dsRNA positive cells was analyzed using the BD Pathway<sup>TM</sup> 435 Bioimaging system in (<b>C</b>) BHK and (<b>D</b>) Huh7, respectively. (Graph is plotted through calculating the percentage of NS1, NS3, and dsRNA expression in each image.) Data indicate the average value of triplicates (mean ± SD). ** <span class="html-italic">p</span> = 0.01; *** <span class="html-italic">p</span> &lt; 0.001, as compared with the control.</p> Full article ">Figure 3 Cont.
<p>Honokiol inhibits dengue virus translation and replication in cells. (<b>A</b>) Immunofluorescence assay of honokiol-treated, DENV-infected BHK cells with MOI = 0.1. The viral NS1, NS3, and dsRNA antigens were detected after 48 h of honokiol treatment; (<b>B</b>) Immunofluorescence assay of honokiol-treated, DENV-infected Huh7 cells with MOI = 10. The viral NS1, NS3, and dsRNA antigens were detected after 48 h of honokiol treatment; (<b>C</b>, <b>D</b>) The percentage of viral NS1, NS3, and dsRNA positive cells was analyzed using the BD Pathway<sup>TM</sup> 435 Bioimaging system in (<b>C</b>) BHK and (<b>D</b>) Huh7, respectively. (Graph is plotted through calculating the percentage of NS1, NS3, and dsRNA expression in each image.) Data indicate the average value of triplicates (mean ± SD). ** <span class="html-italic">p</span> = 0.01; *** <span class="html-italic">p</span> &lt; 0.001, as compared with the control.</p> Full article ">Figure 4
<p>Pre-treatment of honokiol does not affect DENV receptor expression in cells. (<b>A</b>) BHK cells were treated with honokiol at 10 μM concentration for 24 or 48 h and then incubated with dengue virus for 30 min to process the virus attachment. Simultaneously treatment of neutralizing antibodies 137-22 upon infection was used as a control group to block the specific DENV binding. Unbound virus was washed away with PBS and the cells were labeled with anti-DENV E protein antibody and secondary antibody for flow cytomerty analysis; (<b>B</b>) The percentage of cells showing positive fluorescent signals was plotted. Data indicate the average value of triplicates (mean ± SD). *** <span class="html-italic">p</span> &lt; 0.001 as compared with the control.</p> Full article ">Figure 4 Cont.
<p>Pre-treatment of honokiol does not affect DENV receptor expression in cells. (<b>A</b>) BHK cells were treated with honokiol at 10 μM concentration for 24 or 48 h and then incubated with dengue virus for 30 min to process the virus attachment. Simultaneously treatment of neutralizing antibodies 137-22 upon infection was used as a control group to block the specific DENV binding. Unbound virus was washed away with PBS and the cells were labeled with anti-DENV E protein antibody and secondary antibody for flow cytomerty analysis; (<b>B</b>) The percentage of cells showing positive fluorescent signals was plotted. Data indicate the average value of triplicates (mean ± SD). *** <span class="html-italic">p</span> &lt; 0.001 as compared with the control.</p> Full article ">Figure 5
<p>Honokiol may interfere with the endocytotic pathways of DENV entry. Huh7 cells were (<b>A</b>) mock or (<b>B</b>) infected with dengue virus at MOI of 10. The DENV-infected cells were treated with (<b>C</b>) 10 and (<b>D</b>) 20 μM of honokiol for 1.5 h. Immunofluorescence staining was conducted to detect the DENV E protein (green fluorescence) and the early endosome marker, EEA1 (red fluorescence). Numbered side squares represent the magnified images of areas in corresponding panels.</p> Full article ">Figure 6
<p>Honokiol induces a slight cell cycle alteration in Huh7 but not BHK cells. (<b>A</b>) BHK and, (<b>B</b>) Huh7 cells were mock or treated with honokiol for 48 h. The cells were then fixed, stained with PI and analyzed by flow cytomerty. The distribution of cell cycle phases was demonstrated by bar charts. Data indicate the average value of triplicates (mean ± SD).</p> Full article ">Figure 7
<p>Honokiol does not alter the activation of NF-κB and the expression of IFN-β. (<b>A</b>) Assay of NF-κB activation. Huh7 cells transfected with NF-κB-luciferase reporters were mock or infected with DENV at MOI = 5 and then treated with honokiol at 10 and 20 μM for 48 h, followed by Firefly-Renilla luciferase assay to determine the relative expression of NF-κB responsive luciferase. PMA is an NF-κB activator and was used as a positive control; (<b>B</b>) Assay of IFN-β promoter activity. Huh7 cells transfected with IFN-β-luciferase reporters were mock or infected with DENV at MOI = 5 and then treated with honokiol at 10 and 20 μM for 48 h, followed by Firefly-Renilla luciferase assay to determine the relative expression of IFN-β promoter-driven luciferase. Poly(I:C) is a immunostimulant similar to dsRNA and was used as a control. Data indicate the average value of triplicates (mean ± SD). *** <span class="html-italic">p</span> &lt; 0.001 as compared with the control.</p> Full article ">
3050 KiB  
Review
Structural Conservation and Functional Diversity of the Poxvirus Immune Evasion (PIE) Domain Superfamily
by Christopher A. Nelson, Megan L. Epperson, Sukrit Singh, Jabari I. Elliott and Daved H. Fremont
Viruses 2015, 7(9), 4873-4893; https://doi.org/10.3390/v7092848 - 28 Aug 2015
Cited by 31 | Viewed by 8261
Abstract
Poxviruses encode a broad array of proteins that serve to undermine host immune defenses. Structural analysis of four of these seemingly unrelated proteins revealed the recurrent use of a conserved beta-sandwich fold that has not been observed in any eukaryotic or prokaryotic protein. [...] Read more.
Poxviruses encode a broad array of proteins that serve to undermine host immune defenses. Structural analysis of four of these seemingly unrelated proteins revealed the recurrent use of a conserved beta-sandwich fold that has not been observed in any eukaryotic or prokaryotic protein. Herein we propose to call this unique structural scaffolding the PIE (Poxvirus Immune Evasion) domain. PIE domain containing proteins are abundant in chordopoxvirinae, with our analysis identifying 20 likely PIE subfamilies among 33 representative genomes spanning 7 genera. For example, cowpox strain Brighton Red appears to encode 10 different PIEs: vCCI, A41, C8, M2, T4 (CPVX203), and the SECRET proteins CrmB, CrmD, SCP-1, SCP-2, and SCP-3. Characterized PIE proteins all appear to be nonessential for virus replication, and all contain signal peptides for targeting to the secretory pathway. The PIE subfamilies differ primarily in the number, size, and location of structural embellishments to the beta-sandwich core that confer unique functional specificities. Reported ligands include chemokines, GM-CSF, IL-2, MHC class I, and glycosaminoglycans. We expect that the list of ligands and receptors engaged by the PIE domain will grow as we come to better understand how this versatile structural architecture can be tailored to manipulate host responses to infection. Full article
(This article belongs to the Special Issue Poxvirus Evolution)
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<p>The poxvirus immune evasion (PIE) domain structures adopt a strikingly similar fold despite considerable sequence diversity. Strands in the β-sandwich core domain (cyan) are numbered the same for all four structures to aid comparison. The decorations unique to each structure (dark blue) are labeled (h for helix, β for strand). CPXV203 does not contain a strand β11 in sheet I. Similarly, strand β8 of sheet II is absent from vCCI and A41. The disulfide bonds are labeled in red (<b>A</b>–<b>E</b>). All ribbon diagrams are shown in the same orientation and at the same scale. Structures displayed include: rabbitpox vCCI (2FFK) [<a href="#B20-viruses-07-02848" class="html-bibr">20</a>], cowpox CPXV203 (4HKJ) [<a href="#B28-viruses-07-02848" class="html-bibr">28</a>], vaccinia A41(2VGA) [<a href="#B22-viruses-07-02848" class="html-bibr">22</a>], and ectromelia CrmD C-terminal SECRET domain (3ON9) [<a href="#B25-viruses-07-02848" class="html-bibr">25</a>]. Figure made in PyMol [<a href="#B47-viruses-07-02848" class="html-bibr">47</a>].</p> Full article ">Figure 2
<p>PIE domain connectivity diagrams highlighting the conserved core β-sheet architecture (cyan) and unique connecting decorations (dark blue). The disulfide bonds are labeled in red (A–E). Ligand contact regions are annotated with magenta stars for those PIE domains with structurally defined interactions.</p> Full article ">Figure 3
<p>Structure based sequence alignment for the PIE domains of <a href="#viruses-07-02848-f001" class="html-fig">Figure 1</a>. Numbering of the core β strands (cyan) is given above the sequences. The decorations are indicated (dark blue), with new strands as arrows, helices as cylinders, and extended coil as a blue line above the sequence. The decorations occur as insertions primarily to the β6–β7 loop, the β7–β9 loop, and at the C-terminus. Disulfide bond cysteines are marked above the alignment, with red circles containing letters of the different disulfide-bond pairs (A, B, C, D, or E) as indicated in <a href="#viruses-07-02848-f001" class="html-fig">Figure 1</a> and <a href="#viruses-07-02848-f002" class="html-fig">Figure 2</a>. The contacts made by ligand are marked with stars (magenta) under each sequence.</p> Full article ">Figure 4
<p>Comparison of PIE domain surface properties. The molecules and orientations are the same as in <a href="#viruses-07-02848-f001" class="html-fig">Figure 1</a>. (<b>a</b>) Electrostatic potential surfaces calculated using APBS [<a href="#B52-viruses-07-02848" class="html-bibr">52</a>]. Negative charge in red and positive charge in blue from −3kT/e to +3kT/e. Crystallographically observed contact surfaces for ligand are circled; (<b>b</b>) Sequence conservation within individual families was mapped to the molecular surface and colored magenta for highly conserved and green for variable. Because so few CrmD exist and CrmB and CrmD are closely related, sequences for CrmB and CrmD SECRET domains were aligned and conservation mapped to the CrmD molecular surface.</p> Full article ">Figure 5
<p>PIE domain proteins use unique determinants to engage ligands. vCCI primarily employs sheet II to bind CC-chemokines (2FFK) [<a href="#B20-viruses-07-02848" class="html-bibr">20</a>], CPV203 (T4) uses the edge of the β-sandwich plus part of sheet I to bind MHC class I/peptide complexes (4HKJ) [<a href="#B28-viruses-07-02848" class="html-bibr">28</a>], and CrmD appears to use sheet I for the binding of a low-affinity chemokine (3ON9) [<a href="#B25-viruses-07-02848" class="html-bibr">25</a>]. All ribbon diagrams are shown with the PIE domain in the same orientation.</p> Full article ">Figure 6
<p>Sequence alignment of representative members of the PIE families. The positions of the vCCI core strands (cyan) are shown above the sequences. Cysteines are boxed in yellow. The predicted disulfide bonds are lettered in red. The predicted signal peptides are shown under the red bar. The PIE family name is given before the ORF name when they differ.</p> Full article ">Figure 7
<p>Dendrogram of PIE domain sequences showing relatedness among representative members of the PIE domain families. The tree is midpoint rooted for purposes of illustration. Values in percent at internal nodes indicate posterior probabilities calculated for the Bayesian inference of phylogeny from the alignment in <a href="#viruses-07-02848-f006" class="html-fig">Figure 6</a> using MrBayes v3.2.0 [<a href="#B81-viruses-07-02848" class="html-bibr">81</a>]. The scale bar relates branch lengths to the number of expected substitutions per site. The family names are shown at the terminal nodes.</p> Full article ">
1250 KiB  
Review
Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways
by Peter Wimmer and Sabrina Schreiner
Viruses 2015, 7(9), 4854-4872; https://doi.org/10.3390/v7092849 - 28 Aug 2015
Cited by 50 | Viewed by 10029
Abstract
Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last [...] Read more.
Posttranslational modifications (PTMs) of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways. Full article
(This article belongs to the Special Issue Viruses and the Ubiquitin/Proteasome System)
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Figure 1
<p>Overview of some viruses that modulate host ubiquitinylation pathways. Listed are viruses known to encode proteins exhibiting functions of cellular regulatory proteins involved in ubiquitinylation. Described in detail in the text.</p> Full article ">Figure 2
<p>Overview of some viruses that modulate host SUMOylation pathways. Listed are viruses known to encode proteins exhibiting functions of cellular regulatory proteins involved in SUMOylation. Described in detail in the text.</p> Full article ">
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