Virus = Genetic entities (RNA of DNA), protected by a protein coat (and sometimes an envelope (lipid membrane +

glycoproteins)), which upon infection of a suitable host controls the synthesizing system of the cell in such way that new viruses
are produced, often coinciding with
visible pathological effects on the cell and the organism

 causal agents of many infectious diseases

 selfish set of genes (2-900 genes; 2000 - 106 basepairs
 only 2 smaller known pathogens:
o viroids
o prions
 Main properties:
o Infectivity: property to infect a cell, to multiply, and to leave the cell
o Stable survival in extracellular space
o Obligatory intracellular parasites
 Positive vs negative strand:


 Forms:
o Rod-shaped: based on helical symmetry, not circular symmetry
o Isometric: based on icosahedron, which is the lowest-energy configuration for the
degrees of symmetry it offers -> isotropic interactions of particles with the surface
o Complex
o Pleiomorphic
 Triangulation (T): all isometric viruses are icosahedral, yet their size (and further triangulation
of their surfaces) can be variable depending on their genome size

 Infection cycle:
 Replication timeline


 Classification/taxonomy:

 Segmented vs non-segmented


 Baltimore classification:


 Infectivity:
 Most viruses are (-) ssRNA viruses

 Proteins encoded by viruses

 Forces driving its evolution:

o Mutation:
 Higher mutation rate compared to cellular DNA due to the lack of
proofreading-ability in RNA polymerases -> RNA viruses evolve 10000-
100000 times faster than their hosts
 Technically viruses are quasi-species

 

o Recombination:
 of viral gene (sets) and/or between viral and cellular RNAs
 recombination in nature is rare
 constraints:


o (Segment) reassortment
 o
 low population passages under selective pressure may lead to lower fitness


 Serotype?
 How to detect functional viral RNAi suppressor:
o agroinfiltration of GFP-virus expression construct
 Green fluorescing spots (and/or HR depending on the plants R genes) after
agroinfiltration -> silencing effective
 No spots -> silencing not suppressed
o In vivo labeling with 35 S -Met
 No host protein synthesis -> silencing effective
 Host protein synthesis -> silencing not effective
 Plant viruses:

 ss(+)RNA accounts for 77% of all plant viruses

o since ss(+)RNA can be translated directly into proteins
 plant cells only translate monocistronic messengers (mRNAs) with one open reading frame
(ORF). Adaptation in plant viruses to circumvent this bottleneck:

Protein split into

multiple small proteins
 growth cycle:
o replication
o movement (movement protein)
o encapsidation (coat protein(s))
 RNA viruses of plants evolutionary related to RNA viruses of animals
o Monophyletic based on helicase, polyphyletic based on polymerase
o By recombination

plant (+)ssRNA virus:

 infection cycle:

uncoating

recruitimen
t

 Entrance:
o Piercing (by herbivore or biting vector)
 o Mechanical damage
o Unlike animal viruses, no receptor-mediated endocytosis
 Replication: in cytoplasm

 Form replication complexes (RCs): modified cellular membranes, viral RNA and viral replicase
proteins
 RNA silencing/RNA interference (RNAi)


oSuppressed at different steps by viral suppressor proteins

oEffective silencing also suppresses host transcription, to:
 Increase resources available for viral protein synthesis
 Block IFN viral pathways
 Movement through plasmodesmata (PD)
 

Picornavirus:
 Characteristics:


 Examples:
o Hepatitis A
o Rhinovirus (common cold)
o Poliovirus (poliomyelitis; human enterovirus C)
o FMDV
 Very large virus family
o 5 subfamilies
o 2 floating genera
 Are grouped as an order based on
o Shared characteristics (virus particles, genome characteristics, translation strategy)
 T=1 (or pseudo T=3)
  (+)ssRNA
 Often with genome-linked viral protein (VPg) at 5’end and 3’ polyA tail
 RNA is not capped -> VPg attached to 5’ end
 Helicase-protease-RDRP cassette

 Polyprotein processing
o Phylogeny
 Translation strategy:
o Typically one large ORF
 Some 2 ORFs
 Some segmented, with 1 ORF per segment
o Translation into single large polyprotein
 eIF4G is cleaved yet still can bin to IRES and initiate translation -> prevents
cap-dependent translation of host mRNAs -> cap-independent
 Internal ribosome entry site (IRES) in 5’ noncoding region (NCR)

 Attenuation due to point mutations in IRES -> reduced translation

efficiency
o Processed into individual proteins
 By proteases
 

 3C protease is conserve in all picornaviruses:



o By ribose-skipping elements
 Co-translational codon skipping
 Ribosome moves to next codon but does not form peptide bond -> 2-protein
 Looks like cleavage; one ORF results in 2 proteins
 Functions of proteins:
 Role of capsid:

 Receptors:
o Glycoproteins
o Receptor binding site in canyons -> antibodies cant reach -> escape immune system

o VPg functions as primer during RNA replication

o Translation is prevented and instead replicative mode of RNA in induces by binding
of 3CD with poly-rC binding protein (PCRBP2) to the 5’-UTR (IRES)
 Shut off the host protein synthesis:
 

Bunyaviridae:
 vector: anthropods (propagative), rodents
o has little effects on invertebrate host
o transmission:
 sometimes transovarial (offspring)
 sometimes propagative
o envelope glycoproteins are vector determinants and are required for receptor
binding and subsequent infection

 Examples
o Rift valley fever virus(RVFV)
o CCHF
 Can infect both plants and animals
o plant-infecting bunyaviruses differ by the additional presence of a separate ORF
coding for the cell-to-cell movement protein (to transmit via plasmodesmata)
 Characteristics:
o Spherical, enveloped (80-120nm)
 o (-)ssRNA
o Ambisense: non-overlapping gene ORF can be on original – strand or on + strand
o Segmented tripartite RNA genome (large, medium and small RNA)

To cross plasmodesmata Virulence factors

In plants: suppresses RNAi

In animals: suppresses interferon-induced antiviral defense

o Gc domain contains fusion domain

o
 Conserved 3’-terminus sequences since termini contain promoter sequence for viral genome
transcription-replication
 Complementary 5’ and 3’ end -> pin or pseudo-circularity -> changing accessibility of termini
allows for modification of replication vs transcription rates
 Cap-snatching:


o RNA contains core catalytic polymerase domain

o Facilitates binding of transcription complex to the viral RNA

  Lifecycle:

Membrane fusion (by

fusion domain of Gc)
due to pH change

 Emergence of new viruses by genome reassortment with co-infecting viruses

 Potential in biological warfare as cDNA clone of (+)RNA viruses
o Directly infectious
o Express L and N proteins; and L, M and S RNA
 Vaccines against these viruses can be produced by removing the M RNA and supporting
single round (non-cyclic) infection only

Orthomyxoviridae
 Examples:
o Influenza A
 Characteristics:
o (-) ssRNA
o 8-9 RNA segments
o Transmission: airborne
o Quasi-spherical or filamentous, enveloped (80-120nm)
o Envelope derived from host cell plasma membrane by budding
 Influenza virus:
o Helical nucleocapsid (RNP): 9 copies of NP monomers and 1 copy of polymerase (3
subunits (PA, PB1, PB2))
o
o 5’ and 3’ complementarity
o 3’ terminal sequence differs per genus

o
o Virion structure:
(trimer)

(tetramer)

o
 o

NP, PA, PB1 and PB2

involved in stabilization of
antigenome

PA, PB1 and PB2 are

subunits of the polymerase

NA destroys cell receptors during cell

escape (budding), otherwise the
progeny virus will re-infect the same
cell -> thereby simulates viral spread

o Reproduction cycle:

Receptor-mediated endocytosis
mediated by HA trimers

Fusion with host membrane also mediated by HA

trimers

Uncoating mediated by M2 ion channel proteins

Transcription:

In the nucleus!!  initiated by PB1 and PB2 binding

to 5’ cap (cap-snatching)
 termination by PB1 reaching the
3’ end

Virion formation: alignment of 8 rod-like

structures

o
o Influence virus RdRp associates to the C-terminal domain of RNA polymerase II
 Inhibitors of RNA polymerase II inhibit influenza virus replication
o Difference between Bunyavirus and Influenza virus cap snatching:

o Suppresses interferon system

o Segment-specific encapsidation:
o
o Many inter-segment interactions. Amount of intersegment interactions
differs per strain
o Leads to the incorporation of all individual RNA segments into one particle
o Most groups are virulent
o Infect both humans and birds
o Classification based on surface antigens (NA, HA)
o Vaccines:

o
o Types:

o
o Vaccines will only protect against other influenza viruses with the same
hemaglutinin (H) type
o Drawbacks:
 o
o Alternative to vaccination: antiviral drugs

o
o Rapid evolution:
o Point mutations -> antigenic drift
o Reassortment and transfer to different species -> antigenic shift
o Pathogenicity depends on glycoprotein/spike proteins (HA and N) as well as the
amino acid composition of proteins produced by the virus

Retroviridae
 RNA -> DNA incorporated into host DNA
o (-)ssRNA -> mRNA
o (+)ssRNA -> (-)ssRNA -> mRNA
o (+)ssRNA -> DNA/RNA hybrid -> dsDNA -> mRNA
 Examples:
o HIV-1 and HIV-2
 Major cause of cancer (20% of cancer cases) by
o transducing viruses : proto-onc genes are captured from the host genome into the
viral genome
o non-transducing viruses: cellular onc genes are activated by insertion?
 Vectors for gene therapy
 Structure:


 ‘’diploid’’ genome of retroviruses is held together by regular base pairing and by basepairing
host-encoded tRNAs
 RNA structure:



Lentiviruses (one of the genuses of Retroviridae) have a number of
 RT:addition to gag-pro-pol-env
genes in

 frameshift translation of gag/pol promoted by pseudoknot in L-A RNA


 Lifecycle:
 Receptor-mediated entry at low pH (5.5)




 Reverse transcriptase:


 Incorporation into host genome:


 Host increases transport of DNA into the nucleus (by interaction of BAF and Emerin) and
tethering to chromatin (by interaction IN-DNA an Ledgf)
 Dependency of viruses on host cell cycle due to accessibility of DNA) varies per virus
 Gene regulation by
o Splicing
o Polyprotein processing
o Auto/feedback regulation
o Ribosomal frameshifting
 HIV-1:
o core:
o
o GP precursor is cleaved into transmembrane protein and surface protein, which
remain bound by S-S bridges
o Infects CD4+ T-cells
 and lyses them
 by receptor (CD4) mediated endocytosis
 Entry facilitated by transmembrane protein and surface protein
 Leaves the cell by budding
o Hypervariable regions in the surface protein make it hard to produce a vaccine
o Multiple splicings to generate mRNAs for many more (nonstructural) proteins
tat enhances transcription of
provirus sequence -> full-
length transcripts

rev stimulates selective

expression of viral mRNAs
and regulates splicing an
nuclear export of mRNAs

nef reduces host immune

response by removing CD4
and MHC1 receptors from
host cell surface -> virions
can escape
o Antiviral strategies:

o
Pararetroviruses
 dsDNA -> ssRNA -> RNA-DNA -> dsDNA -> mRNA
 do NOT require integration of a genomic DNA copy into the host genome for viral replication!
o However, integrations of (partial) pararetrovirus genome sequences have been found
in many different hosts
 Examples:
o Caulimoviridae (cauliflower mosaic virus)
o Hepatitis B
 Caulimoviridae (cauliflower mosaic virus; CaMV)
o Properties:

o Genome organization

P1-P7
The ribosomal shunt translation
strategy of CaMV has evolved from
ancient Long Terminal Repeats

o Replication cycle

=Structures specialized for aphid

transmission of CaMV

ATF and CPm are aphid transmission

factors

o Reverse transcription:
 o

D1/D2/D3: discontinuities
resulting from purine-rich
sequences (relatively resistant
to RNaseH) -> RNA leftovers
allow for internal priming by RT

 In cytoplasm
 By viral transcriptase (P5)
 Primed by tRNA (meth) on a PBS
o Part of virus is endogenized (Endogenized partial caulimoViral Elements (EVEs)) to
serve the host defense against viruses by a priori activation of RISC into antiviral RISC
(RNAi) -> immunity
 Post-transcriptional gene silencing (PTGS): degradation of target RNAs
 Transcriptional gene silencing (TGS): C-methylation of residues -> inhibition
of transcription
8S is converted into dsRNA and serves as a decoy for CLs (RNAi)

P6 serves as RNAi suppressor protein

 Hepadnaviridae (Hepatitis B virus (HBV))

o Partially dsDNA (rcDNA)
o Major cause of liver cancer
o Dane particle: enveloped nucleocapsid
 o Lifecycle:

NTPC is specifically expressed in liver cells

and acts as receptor for HBV PreS1 region
of the HBV envelope protein binds to NTCP
to facilitate viral entry

o Genome organization:
The overlapping nature of the genome
makes it very susceptible to RNAi: single
siRNAs can reduce all mRNA from ccDNA
but can miss integrated-derived mRNA

o Reverse transcription of pgRNA into vDNA:

o

o Does NOT require splicing for replication, yet splicing occurs and creates splice
variants (function unknown)
o Mode of transmission (horizontal/vertical (perinatal)) differs per HBV genotype
 o No cures (since cccDNA is persistent) and HBV readily develops resistance to most
Nas in monotherapy. Treatments;

Baculoviruses:
 Can be used as biocontrol against lepidoptera
 Insect-specific DNA viruses
 Rod-shaped virions in protein capsules
 Contain polyhedral occlusion bodies (OBs) in the nucleus (nuclear polyhedroviruses)
o Alkali-sensitive
o Mainly composed of polyhedrin
o Virus particles are released in insect midgut
o OB dissolve -> pass through endoperitrophic membrane -> into midgut columnar
epithelial cells -> nucleocapsids move to nucleus where DNA is uncoated and gene
expression/replication starts (primary infection)
 -> progeny virus buding from basal lamina side to infect other cells ->
budded viruses (BV) -> secondary infection
 -> progeny released after host cell lysis after host death (polyhedrin and p10
are hyperexpressed) -> polyhedra -> solubilization in gut -> polyhedral
derived viruses (PDV)
 Per os infectivity factors (PIFs):

 core genes involved in the (exclusively oral) infection pathway

 form a protein complex in ODV envelope (except for PIF5)


 inner nuclear membrane sorting motif

o OB and ODV types are virus species-dependent

o Upon death, occluded viruses can be released to environment -> new infection cycle
 Types:
 4 genera based on set of 37 shared ORFS (core genes), co-evolved with their hosts:

Genus type host Budded virus fusion

protein
alphabaculovirus NPVs GP64 (group I)1 or F
protein2 (group II)
betabaculovirus GVs lepidoptera F protein2
deltaabaculovirus NPVs hymenoptera F protein2
(sawflies)
gammabaculovirus diptera (mosquitos) - (since no BVs)

1= GP64:

o Forms trimers
o Involved in membrane fusion -> syncytium of cells
o Point out from virus to interact with cellular receptors -> endocytosis
o Forms pore -> nucleocapsid (NC) entry into cytoplasm
o NC moves along actin filaments to the nucleus
2= F-protein:
o Forms trimers
o Involved in membrane fusion -> syncytium of cells
o Original envelope protein
 Additionally, alpha- and betabaculoviruses share 62 ORFs
 Related to several other nuclear arhtropod-specific large DNA viruses:
 dsDNA
 circular genome
 90-180 ORFs in both orientations, most of which do not contain introns -> no splicing
 5% small spacer regions
 Gene cassettes as building blocks -> non-overlapping ORFs
 Genes distributed over both DNA strands
 Expressed in a temporal cascade
  Dispersed homologous regions with palindromic repeated elements (hrs)
o Can fold with opposite strand or within own strand -> 3D structures which can
interact with proteins

Parvovirus:

 animal virus
 Small genome (<10kb, 5 proteins, 2 or 3 promoters, replication and viral capsid proteins)
o Differential splicing
o Alternative translation
 linear ssDNA
o have to be made dsDNA before it xan be recognized by RNA polymerase II
o one molecule per virion
o -sense in autonomous viruses, qually distributed + or -sense in dependoviruses
DNA with terminal hairpin
structures needed to
prime DNA replication

NS1:

 Transcriptional activator for all promoters

 Site specific endonuclease (hairpin cleavage)
 ATP binding, ATPase and helicase activity (unwinding
of the hairpins
 Induces cell cycle arrest in S-phase
 Oncolytic properties

NS2: nuclear egress of progeny virions

 Replication strategy:

DNA replicase direction; eliminates need for telomeres; results in dsDNA

molecule with hairpin at one end
 depends on host DNA polymerase and other cellular proteins for transcription an replication
o requires host cell to be in S-phase for replication, since the virus cannoy induce
hostcell S-phase -> parvoviruses can only replicate in dividing cells
o some parvoviruses ue adeno- or herpesviruses as helpers to bring cells in S-phase
o Naked DNA can infect all cell types, but is limited to the S-phase of the appropriate
cells to replicate
 naked icosahedral capsid
Host specificity is mainly determined
o T=1
at the level of DNA uncoating, not
o 60 protein subunits
receptor binding.
 8-stranded antiparallel β-barrel
 Infection cycle:


 Subfamilies:

 Types:

AAV without helper: AAV with helper:

 Hibernate or integrate into host DNA.  E1A: activates cellular transcription factor E2F, which enables
 Helper virus can still help with the integration promoter
into host DNA/excision of viral DNA, and  E1B and E4: transport mRNAs into cytoplasm
production of virus progeny.  VA-RNA: acts as decoy -> prevents phosphorylation of initiation
factor eIF2α which would lead to inhibition of translation
  Some parvoviruses can integrate site-specifically in the host genome
 Since parvoviruses typically prefer fast-replicating cells, they prefer tumor cells over normal
cells and therefore can be used for tumor treatment.

Baculovirus and parvovirus-based biotechnology:

 Parvovirus:
o can be used as gene delivery vector for gene therapy
o AAV strains can be used to integrate into host DNA,
 Pros:
 Safe
 Replication deficient
 Perfect for treating single-gene deficiencies
 cons:
 integrtion is rare
 depends on strain
 strains differ in tissue tropism and pre-existing immunity
 limited packaging capacity
 high doses needed for whole-body treatment
 method:
 triple plasmid infection


 Production in insect cells with baculovirus expression systems

P10 or polyhedrin
e.g. GFP
(ph) promoters are
used to drive foreign
gene expression

 Pros:
  Cons:
o baculoviruses become too large for direct cloning -> can be
circumvented by
 replacing viral ORFs through homologous
recombination (including transposition) or
restriction digestion
 both methods require various rounds of
selection
o most likely candidates: application:
 pathogen surface prorteins vaccines/diagnostics
 nucleoproteins diagnostics
o application: vaccines and diagnostics
 baculovirus:
 VP80 Is required for ODV formation
(migration of NCs from virogenic
stroma to nuclear periphery to forms
BVs and later ODVs.

VP80 is NOT require for DNA

replication

o Baculovirus as mammalian transfer-vector BAC-Mam system

 o Applications:
 Glycoproteins, subunits and (e)VLPs
 Surface display, antigen carrier
 Viral vectors
 Gene delivery

Megavirales (giant viruses):

 dsDNA
 phylogeny: nucleocytoplasmic large DNA viruses (NCLDVs)
 contain genes not identified before in a viral genome, and are the hallmark of cellular
organisms -> fading boundary with ‘’life’’

 Can be infected by other viruses (which in turn are large)

o Provirophages
o Transpovirons
 Mobile genetic elements (MGEs; mobilome) of giant viruses contains provirophages and
transpovirons
o Transposable elements
 o Self-splicing elements
o Plasmids
o Viruses
 Model for eukaryote nucleus:


overview