Role of Actin Microfilaments in Black Creek Canal Virus Morphogenesis

Cell culture and virus strains.

Vero and BHK21 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and supplemented with penicillin-streptomycin antibiotic mix. Once confluence was reached, the BHK21 cells were maintained in DMEM with 5% FBS and the Vero cells were maintained in DMEM with 2% FBS. The stock of BCCV (3 × 10⁷ PFU) was grown and titered on Vero E6 cells. Vesicular stomatitis virus (VSV) (5 × 10⁹ PFU) was grown in BHK21 cells and plaque purified two times, and titers were determined by plaque assay on BHK21 cells. Punta Toro (PT) virus was grown on Vero E6 cells, and titers were determined by plaque assay.

Reagents.

Monoclonal antibody (MAb) GB04-BF07 recognizing the BCCV N protein was provided by Michael D. Bowen (Centers for Disease Control and Prevention, Atlanta, Ga.). Anti-BCCV rabbit immune serum was a generous gift from Thomas Ksiazek (Centers for Disease Control and Prevention). Nocadozole, cytochalasin D, rhodamine-conjugated phalloidin, and anti-β-actin and anti-β-tubulin MAbs were purchased from Sigma (St. Louis, Mo.). [³⁵S]methionine-cysteine labeling mix was obtained from Amersham Corp. (Arlington Heights, Ill.). Fluorescein isothiocyanate-conjugated anti-mouse and anti-rabbit antibodies were purchased from Southern Biotechnology Associates (Birmingham, Ala.). Sindbis virus expression vectors SIN-rep5 and helper 5′-tRNA were provided as gifts by C. Rice. Hyperimmune mouse ascitic fluid to PT virus was a gift from J. F. Smith and D. Pifat (U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, Md.).

Virus infectious-center assay.

Since plaque assay of hantavirus infections is difficult, we developed an infectious-center assay to measure the virus titers. Tenfold dilutions of BCCV containing 100 μl of medium were placed on monolayers of Vero E6 cells grown in six-well plates, and virus was adsorbed for 2 h at 37°C. The cells were rinsed once in phosphate-buffered saline (PBS) and supplemented with 3 μl of liquefied 1% methylcellulose in DMEM with 2% FBS. After 6 days of incubation at 37°C, the methylcellulose-containing medium in the wells was liquefied by placing the plate on ice for 20 min and removed. The cells were rinsed three times in cold PBS to remove any residual methylcellulose and fixed in chilled 95% ethanol with 5% acidic acid for 20 min at −20°C. Nonspecific binding was blocked by preincubating the fixed cells in PBS containing 3% bovine serum albumin (BSA). The cells were then incubated with a 1:500 dilution of anti-N MAb. The bound antibody was detected with secondary antibody conjugated with horseradish peroxidase by using an immunostaining kit from Vector Laboratories (Burlingame, Calif.). The infectious centers were observed under a light microscope and counted.

Construction of recombinant plasmids.

In our previous studies, we had cloned the entire sequences of BCCV S and M segments into a TA cloning vector, pCR 2.1, and used those plasmids (pCR-N, pCR-GPC) for nucleotide sequence analysis of the BCCV N and GPC genes (24). In the studies described here, the coding sequences of BCCV N and GPC genes were amplified by PCR from pCR-N and pCR-GPC plasmids with specific primers linked with XbaI adapters. The PCR fragments were digested with XbaI overnight at 37°C and purified through an agarose gel with glass beads (24). SIN-rep5 expression vector was linearized with XbaI and dephosphorylated by treatment with alkaline phosphatase according to the manufacturer’s instructions. The ligation reaction was carried out at 12°C overnight, followed by transformation of Escherichia coli. The orientations of the cloned DNA inserts were determined by restriction digestion analysis, based on restriction maps of the vector and the DNA insert sequences (4, 24). Transcription reactions, electroporation, and handling of both the recombinant and the helper Sindbis virus RNA synthesized in vitro from linearized plasmid constructs were performed as described by Brebenbeek et al. (4). Expression of the recombinant proteins was carried out in BHK21 cells and examined by both indirect immunofluorescence analysis (IFA) and radioimmunoprecipitation analysis.

IFA.

Cells grown on coverslips were rinsed three times in PBS and fixed in 3.7% paraformaldehyde for 15 min at room temperature. The fixed cells were then permeabilized in 0.1% Triton X-100 for 5 min. To eliminate nonspecific binding, the cells were preincubated with PBS with 3% BSA for 30 min at room temperature in a humidified chamber. Antibody dilutions of 1:100 for anti-BCCV sera, 1:500 for anti-N protein MAb, and 1:50 for phalloidin were prepared in PBS containing 3% BSA and added to the permeabilized cells for 30 min, with incubation at room temperature. After washing, anti-BCCV protein bound antibody was detected by incubation with anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody for 30 min at room temperature. Actin microfilaments were detected with rhodamine-conjugated phalloidin added to the secondary antibody. Coverslips were washed three times with PBS, mounted with Vectashield (Vector Laboratories, Burlingame, Calif.), and examined with a Nikon Optiphot microscope.

Coprecipitation of BCCV N protein with actin.

Three days postinfection, BCCV-infected Vero cells grown in 35-mm-diameter dishes were rinsed in cold PBS three times and lysed in 500 μl of lysis buffer (10 mM Tris HCl [pH 7.5], 1 mM EGTA, 100 mM NaCl, 0.5% Triton X-100) for 15 min on ice. In parallel, the same steps were carried out with BHK21 cells transfected with recombinant Sindbis virus RNAs expressing BCCV N protein two days posttransfection. The lysed proteins were separated from cell debris by centrifugation and then supplemented with either 1 μl of anti-β-actin MAb or 1 μl of anti-β-tubulin MAb and placed on ice for 1 h. Subsequently, 20 μl of Sepharose beads conjugated with protein A was added, and the tubes were left rotating overnight at 4°C. The Sepharose beads were collected by centrifugation and washed three times in the lysis buffer. The bound proteins were dissociated from the antibody by boiling in the Laemmli sample buffer for 5 min and separated on 10% polyacrylamide gels (14). Then the separated proteins were transferred to a nitrocellulose filter (Bio-Rad, Hercules, Calif.). The transferred BCCV N protein was detected by Western blotting with an ECL kit (Amersham, Little Chalfont, Buckinghamshire, England) and anti-N MAb.

Radioimmunoprecipitation analysis.

Virus-infected cells were washed once in PBS supplemented with Eagle’s medium without methionine and cysteine and incubated for 30 min at 37°C. Cells were then labeled with [³⁵S]methionine-cysteine labeling mix (100 μCi) for 3 h at 37°C and chased for 15 min. Cells were washed with ice-cold PBS and lysed with 600 μl of lysis buffer (10 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 1% Triton X-100, 20 mM EDTA [pH 8.0]). Nuclei and cell debris were removed by centrifugation at 12,000 × g for 10 min. Cell lysate then was combined with 1 μl of anti-BCC virus rabbit sera by rotating for 1 h at 4°C. The antigen-antibody complexes were precipitated by incubation with 20 μl of protein A-Sepharose (Boehringer Mannheim, Indianapolis, Ind.). The precipitates were pelleted by centrifugation, washed three times with cold lysis buffer, and resuspended in Laemmli sample buffer (14). The samples were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Colocalization of BCCV N protein with actin filaments.

Immunofluorescence staining of BCCV-infected Vero cells with a specific anti-BCCV rabbit serum elicits a characteristic filamentous pattern that is strikingly reminiscent of that of actin microfilaments. At 3 days postinfection, approximately 40% of the infected cells demonstrated this pattern (Fig. 1A). The fraction of infected cells showing the filamentous pattern increased, up to 80%, by 6 days postinfection (Fig. 1C). It seemed likely that the observed pattern was the result of interaction of the BCCV proteins with cytoskeletal structures. To examine this possibility, the BCCV-infected cells were subjected to treatment with cytoskeleton-disrupting drugs and examined by IFA. Treatment of the BCCV-infected cells with cytochalasin D, a microfilament-disrupting drug, resulted in a complete disintegration of the observed filamentous structures (Fig. 1B). In contrast, incubation of the infected cells in the presence of nocadozole, a microtubule-dissociating drug, did not have any effect on these structures. To gain further evidence of actin microfilament involvement, the BCCV-infected Vero cells were examined by double immunofluorescence staining with a specific anti-N protein MAb and phalloidin to stain actin microfilaments. As shown in Fig. 1E, the filamentous antigen pattern in infected Vero cells, observed predominantly at the edge of the cells, was found to be colocalized with the actin microfilaments stained with phalloidin (Fig. 1F). Thus, these results strongly indicate the association of the BCCV N protein with actin microfilaments.

BCCV nucleocapsids and N proteins preferentially bind to the actin microfilaments.

The BCCV genome encodes three major proteins: the G1 and G2 glycoproteins are derived from the GPC and are localized in both the Golgi complex, as inferred from studies with other members of the Bunyaviridae family (9, 16, 23), and the plasma membrane (8, 25); N protein is expressed as a cytoplasmic protein and is assembled into nucleocapsids. To examine the possible interaction of BCCV G1 and G2 proteins with actin microfilaments, the coding sequences for the GPC and N proteins were expressed separately with the Sindbis virus expression system. BHK21 cells were electroporated with the recombinant self-replicating RNAs, and the synthesis of the BCCV proteins was examined by radioimmunoprecipitation assay. Figure 2 shows that the G1 and G2 glycoproteins are synthesized at similar levels and processed to sizes which correspond to those of the viral proteins in infected cells. At 24 h posttransfection, BHK21 cells expressing BCCV proteins were fixed and examined by IFA (Fig. 3). The BCCV glycoproteins expressed in BHK21 cells can be observed both in the perinuclear region and in the endoplasmic reticulum (Fig. 3A). The proteins appear to be distributed throughout the cytoplasm, showing no structures reminiscent of those of actin microfilaments (Fig. 3B). In contrast, BHK21 cells expressing the N protein exhibit the filamentous pattern (Fig. 3C) like that observed in BCCV-infected cells and colocalized with actin filaments (Fig. 3D). However, the filamentous pattern in BHK21 cells is not as prominent as that observed in BCCV-infected Vero cells, and the number of cells showing this pattern was much lower (5 to 10% at 2 days postinfection). Taken together, these results indicate that the BCCV N protein, either expressed alone in transfected cells or presented in the form of a ribonucleoprotein complex (RNP), is capable of binding to the actin filaments.

BCCV N protein binds to actin monomers.

In living cells, actin is present in two forms: G (monomeric) and F (filamentous). To confirm the immunofluorescence results and elucidate whether BCCV N protein and BCCV RNP are capable of binding to the G form of actin, we performed coimmunoprecipitation analysis of the BCCV N protein with anti-β-actin antibody (Fig. 4). In parallel, we carried out the same procedure with anti-β-tubulin antibody. BCCV-infected Vero cells at 3 days postinfection and BHK21 expressing the BCCV N protein alone at 2 days postelectroporation were compared. At selected time points, the levels of N protein in both cell types were found to be similar, as judged by IFA. During lysis at 4°C, only soluble G actin was extracted by the lysis buffer applied to the cells. Figure 4 shows that the N protein is coimmunoprecipitated with actin from both the infected Vero and the electroporated BHK21 cells. In contrast, no signal was obtained with anti-β-tubulin antibody. Coimmunoprecipitation of the N protein from BCCV-infected Vero cells was more efficient than coimmunoprecipitation from the electroporated BHK21 cells, which is consistent with the IFA observations showing less extensive association of N protein with actin filaments in BHK21 cells. In addition, we examined the Western blot of coimmunoprecipitated BCCV proteins with anti-BCCV rabbit immune serum, which recognizes all the viral proteins. No BCCV glycoproteins were detected on the blot; the only band seen on the gel was the one corresponding to the N protein, indicating that the BCCV glycoproteins are not coprecipitated with the actin antibody (not shown). From these data, we conclude that the interaction between the BCCV N protein and actin is not limited only to the filamentous form of actin, but that soluble G actin is also involved.

Depolymerization of actin microfilaments inhibits BCCV release.

Although the association of viral proteins with actin microfilaments has been documented for many viruses, not all of the reported viruses exhibit dependence on this cytoskeletal structure. For example, VSV and influenza virus matrix proteins bind actin microfilaments efficiently (2). However, treatment of either VSV- or influenza virus-infected cells with cytochalasin D does not affect the yield or release of these viruses, indicating that actin is not critical for VSV or influenza virus morphogenesis (10, 11, 27). Therefore, we were interested in examining whether the interaction of the BCCV N protein with actin microfilaments is of any significance for viral morphogenesis or release. Vero cells were infected with BCCV (at a multiplicity of infection [MOI] of 1) and at 3 days postinfection supplemented with medium containing various concentrations of cytochalasin D. The medium was collected after 24 h, and the released-virus titers were quantitated by infectious-center assay. Figure 5A shows that the release of BCCV is significantly reduced in the cells with disrupted actin microfilaments. Less than 1 μg of cytochalasin D per ml was sufficient to induce the inhibitory effect. At this concentration, the released-BCCV titer decreased from 2.0 × 10⁶ to 5.9 × 10⁵ infectious-center units (ICU)/ml. In cells treated with higher cytochalasin D concentrations, progressively less BCCV was released. At 3 days postinfection, in the infected cells supplemented with 25 μg of cytochalasin D per ml, the virus titer decreased to 6% relative to the yield from uninfected cells. As expected, treatment of VSV-infected cells with cytochalasin D (Fig. 5B) did not affect the release of the virus, which is consistent with previous observations (27).

To ascertain whether the inhibitory effect of disruption of actin filaments is unique for hantaviruses, we also examined the effect of cytochalasin D on PT virus, a member of the Phlebovirus genus of Bunyaviridae which is assembled by budding in the Golgi complex. Vero E6 cells were infected at an MOI of 1; at 3 days postinfection, the medium was replaced with medium containing 25 μg of cytochalasin D per ml, and after 24 h the released-virus titers were determined by infectious-center assay. The result of the assay showed no significant change in the titers of PT virus released into the medium (5.0 × 10⁶ ICU/ml with cytochalasin D and 4.8 × 10⁶ ICU/ml without), indicating that the disruption of actin filaments does not affect the yield of released PT virus. Therefore, the inhibitory effect of cytochalasin D is not observed with a member of a different genus of the Bunyaviridae.

There are a number of viruses that have been documented to employ phagocytosis as a mode of entry into the host cell (7). The phagocytosis depends on actin microfilaments. Thus, it is conceivable that the cytochalasin D inhibitory effect on BCCV release was a consequence of events at the level of virus entry, which by itself does not require the interaction of BCCV N protein with actin microfilaments. To investigate the possible effects on early stages of infection, we quantitated the virus titers in medium from cells that were pretreated with cytochalasin D for 30 min and then infected with BCCV in the presence of this drug for 2 h. As shown in Fig. 6, none of the cytochalasin D concentrations used in the experiment were found to affect BCCV yields. A slight decrease of BCCV titers was observed at higher concentrations and is likely to be due to a loss of a fraction of cells from the fragile monolayer caused by the drug during virus adsorption. Since treatment with cytochalasin D is not known to inhibit either mRNA or protein synthesis, it is unlikely that the decrease in the extracellular yield of BCCV was due to an inhibition of viral protein synthesis. Neither does the drug interfere with transport of the BCCV glycoproteins to the cell surface, the proposed site of BCCV assembly in epithelial cells (25), as determined by cell surface staining (data not shown).

The inhibition by cytochalasin D could occur either at the level of the virus assembly or during virus release from the plasma membrane. To determine in which of these stages the interaction of BCCV N protein with actin microfilaments might be involved, we examined the ratios of extracellular- to cell-associated-virus yield in both cytochalasin D-treated and untreated cells. Table 1 shows that compared to results for untreated BCCV-infected cells, titers of both extracellular and cell-associated BCCV decrease progressively as the amount of cytochalasin D added to the medium increases. However, the cell-associated BCCV titer appears to be more sensitive to the drug treatment than the released-virus titer. At a cytochalasin D concentration of 1 μg/ml, the ratio of extracellular to cell-associated virus is 0.10; when 25 μg/ml is used, the ratio becomes 0.05. This suggests that actin microfilaments are involved in BCCV morphogenesis at the stage of virus assembly.