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Journal of Virology, September 2004, p. 9064-9072, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9064-9072.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

trans-Complementation Allows Recovery of Human Respiratory Syncytial Viruses That Are Infectious but Deficient in Cell-to-Cell Transmission

A. G. P. Oomens and Gail W. Wertz*

University of Alabama School of Medicine, Birmingham, Alabama

Received 9 January 2004/ Accepted 17 March 2004


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ABSTRACT
 
Human respiratory syncytial virus (HRSV) expresses three transmembrane glycoproteins: small hydrophobic protein SH, attachment protein G, and fusion protein F. The genes encoding SH and G can be deleted from the HRSV genome and infectious virus recovered. In contrast, HRSVs lacking the F gene or a functional replacement thereof have not been reported. To generate a system with which to study the roles of the viral transmembrane glycoproteins, including F, in the HRSV life cycle, we generated a cell line expressing a heterologous viral glycoprotein for complementation of glycoprotein function in trans. We previously demonstrated that the baculovirus GP64 protein or a chimeric form of GP64 carrying the 12 C-terminal amino acids of the HRSV F protein (GP64/F) can efficiently mediate HRSV infectivity and improve its stability, when expressed from an engineered HRSV genome. Here, we report the development of a stably transfected Vero cell line (Vbac) constitutively expressing the GP64/F protein. From the Vbac cell line, viruses that lacked the SH and F open reading frames (ORFs) or the SH, G, and F ORFs could be recovered from cDNAs. These viruses, designated RS{Delta}SH,F and RS{Delta}SH,G,F, respectively, had place-keeper ORFs inserted in place of the deleted ORFs to maintain authentic transcription levels. In the Vbac cell line, RS{Delta}SH,F and RS{Delta}SH,G,F could be amplified to near wild-type-level titers, and the resulting viruses were infectious to Vero and HEp-2 cells. After entry into Vero or HEp-2 cells, however, neither virus RS{Delta}SH,F nor virus RS{Delta}SH,G,F was able to spread in the infected cultures. Growth analyses showed that infectious virions were not produced in Vero or HEp-2 cells infected with RS{Delta}SH,F and RS{Delta}SH,G,F. Combined, these data provide direct evidence that HRSV F is an essential viral protein required for cell-to-cell transmission and demonstrate that complementation with the GP64 protein in trans constitutes a powerful tool for the study of the role of individual HRSV transmembrane glycoproteins in virus assembly, morphogenesis, and pathogenesis. In addition, the ability to generate infectious but nonspreading viruses may provide an alternative approach for the development of safe and stable HRSV vaccine candidates.


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INTRODUCTION
 
Human respiratory syncytial virus (HRSV) is a pneumovirus classified within the Paramyxoviridae family. HRSV is a major cause of severe lower respiratory tract disease in infants, children, immunosuppressed individuals, and the elderly, and a vaccine is not yet available (4, 7, 20). The 15,222-nucleotide, negative-sense, single-stranded RNA genome of HRSV contains 10 genes encoding 11 known proteins, 3 of which have been characterized as transmembrane glycoproteins: SH, a small hydrophobic protein of unknown function; G, a mucinous protein thought to provide an attachment function; and F, a membrane fusion protein that is responsible for the syncytia observed in infected cell cultures (11, 14, 28). The requirements of the SH and G proteins for HRSV infectivity have been examined in some detail. A cold-adapted HRSV was isolated in Vero cells and found to have a large deletion that included most of the SH and G genes (12). Furthermore, recovery of virus from an engineered cDNA lacking the SH gene showed that the absence of SH did not affect the ability of HRSV to replicate in cell culture and only minimally impacted the yield of infectious virus in vivo (3, 25, 29). In contrast, HRSVs lacking the G gene (which could be recovered in Vero cells) were significantly impaired in their ability to replicate in most cell types and in mice, cotton rats, and humans, indicating that the G protein plays an important role both in vitro and in vivo (25, 27). In a helper virus-dependent mini-replicon system, the F protein was found to be the minimum glycoprotein requirement for production of particles (26). This finding appears consistent with the fact that the F protein induces membrane fusion, a function believed to be essential for HRSV entry. F has also been shown to bind host cell surface molecules, such as glycosaminoglycans, and rhoA, and recently, the F2 subunits of HRSV and bovine respiratory syncytial virus (BRSV) were shown to be determinants of species-specific virus entry into cells (5, 19, 22). Given the above findings, it is not unexpected that viruses lacking the F gene or a functional equivalent have not been described. Glycoprotein substitution studies indicate that the G and F proteins can, in cell culture, be functionally replaced by a variety of heterologous transmembrane glycoproteins, including the BRSV G and F proteins, the bovine parainfluenza virus HN and F proteins, the vesicular stomatitis Indiana virus (VSIV) G protein, and the GP64 protein of baculovirus Autographa californica multiple nucleopolyhedrovirus (2, 17, 18, 24). However, due to the expression of a heterologous replacement protein gene from within the engineered HRSV genome, viruses with glycoprotein gene substitutions cannot be used to directly test the requirement for the HRSV proteins in question.

In order to generate a system with which to study the roles of the viral transmembrane glycoproteins in HRSV infectivity and pathogenesis and to directly test whether the F protein is essential for the propagation of HRSV, we generated a cell line expressing a heterologous viral glycoprotein for complementation in trans. GP64, the major envelope protein of baculovirus Autographa californica multiple nucleopolyhedrovirus, is an excellent candidate for the generation of such a cell line, because (i) it contains both attachment and membrane fusion functions within one protein; (ii) it significantly enhances the stability of HRSV infectivity when it is used as a replacement for the SH, G, and F proteins by expression from within the genome; (iii) it can be expressed efficiently from promoters recognized in the cell nucleus; and (iv) in contrast to the VSIV G protein-expressing cells, GP64-expressing cells show no signs of cytopathic effect (1, 9, 13, 18). In the present report, we describe the generation of a cell line constitutively expressing a chimeric form of the GP64 protein and the use of this cell line to recover from cDNA HRSVs lacking all or two of the three HRSV transmembrane glycoproteins. The resulting engineered viruses allowed us to examine the ability of the virus to spread in Vero and HEp-2 cell cultures in the absence of the F protein or a functional replacement. The results of this study indicate that the complementation approach constitutes a powerful system with which to study the role of the HRSV transmembrane glycoproteins in the biology of the virus and to evaluate the use of infectious, nonspreading HRSVs for the generation of safe vaccine candidates.


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MATERIALS AND METHODS
 
Cells and Abs. Vero 76 (Vero) and HEp-2 cells were acquired from the American Type Culture Collection and grown in standard growth medium containing 3% (for Vero cells) or 5% (for HEp-2 cells) fetal bovine serum. Vero cells were used for the generation of a cell line (Vbac) expressing chimeric glycoprotein GP64/F (see below). Monoclonal antibodies (MAbs) 6 and 19 were provided by Geraldine Taylor (Institute for Animal Health, Compton, United Kingdom), MAb L9 was provided by Ed Walsh (University of Rochester School of Medicine, Rochester, N.Y.), and MAbs AcV1 and -5 were provided by Gary Blissard (Boyce Thompson Institute, Cornell University, Ithaca, N.Y.). The anti-VSIV G Ab was a mouse ascitic fluid acquired from the American Type Culture Collection. The anti-vaccinia virus Ab was a polyclonal serum harvested from rabbits infected with vaccinia virus.

Production and characterization of a cell line expressing a chimeric GP64 protein (Vbac). An open reading frame (ORF) expressing chimeric protein GP64/F was constructed previously (18) and cloned into the expression vector pRc/CMV (Invitrogen). The resulting plasmid (pRc-GP64/F), expressing both the GP64/F and a neomycin resistance gene, was transfected into Vero cells at passage 5 by using Lipofectamine 2000 (Invitrogen). Cells were incubated at 33°C. The following day, transfected cells were detached and plated in six-well plates at various densities. At 48 h posttransfection, 1 mg of G418 sulfate (Geneticin; Invitrogen)/ml was added to the medium, and cells were further incubated at 33°C. G418-resistant colonies were selected between 18 and 34 days later, expanded, and tested for GP64/F expression by using anti-GP64 MAb AcV5 and indirect immunofluorescence (IF). A number of GP64/F-positive colonies were subjected to two rounds of subcloning by plating detached cell suspensions at 1 cell/well in 96-well plates, with continued incubation at 33°C in medium containing G418 sulfate. Following the two rounds of subcloning, a final GP64/F-positive cell line (116-25-1) was selected and designated Vbac, passage 1. Throughout this work, Vbac cells were maintained in standard growth medium containing 5% fetal bovine serum, 1 mg of G418 sulfate/ml, and 25 mM HEPES at 33°C. For characterization of the Vbac cell line, cells were attached to glass coverslips, chilled, and incubated with MAb AcV1 diluted 1:20 in phosphate-buffered saline (PBS)-0.1% bovine serum albumin for 1 h on ice. Unbound Ab was removed by four washes with cold PBS, and cells were fixed with cold 4% paraformaldehyde (PF) for 10 min and then transferred to room temperature; and incubated another 15 min in 4% PF. Cells were washed; blocked with a solution containing PBS, 2% bovine serum albumin, and 0.1% cold-water fish skin gelatin for 1 h at room temperature; and incubated with a goat anti-mouse Ab carrying an Alexa-594 fluorescent conjugate (Molecular Probes) for 50 min at room temperature. Cells were washed with PBS, stained for 10 min with Hoechst reagent (Molecular Probes) to visualize nuclei, mounted, and photographed on an Olympus IX70 inverted microscope. For flow cytometry, Vbac cells were detached in 5 mM EDTA, chilled, and incubated with Abs as described above (without treatment with Hoechst reagent). After the last wash, cells were resuspended in 500 µl of PBS and analyzed in a FACSCalibur flow cytometer (Becton Dickinson), using 50,000 events per sample.

Construction of cDNAs and recovery of infectious HRSVs by using the Vbac cell line. Engineered cDNAs were generated based on a cDNA of the A2 strain of HRSV, which was constructed as described previously (17, 18). Briefly, by using standard cloning techniques, two shuttle vectors containing three ORFs were constructed. For the generation of virus RS{Delta}SH,G,F, the shuttle vector contained the ORFs encoding marker proteins enhanced with green fluorescent protein (GFP) (Clontech), chloramphenicol acetyltransferase (CAT), and ß-glucuronidase (GUS). For the generation of virus RS{Delta}SH,F, the shuttle vector contained the ORFs encoding GFP, HRSV G, and GUS. The translation context of the HRSV G gene in this shuttle vector was altered to enhance the ratio of the amount of G protein anchored to the membrane to the amount of G protein secreted (10, 21); by PCR and conventional cloning techniques, the three nucleotides preceding as well as the first nucleotide following the G start codon were modified to read ACC-ATG-G. This modification resulted in the change of the second amino acid of the G protein from serine to alanine. Surface expression of the altered G protein and its ability to incorporate into virus-induced filaments were confirmed by confocal microscopy (data not shown).

The ORFs contained in each of the shuttle vectors were separated by authentic HRSV intergenic junctions and flanked by the unique restriction sites FseI and AscI. These shuttle vectors were cloned into a cDNA backbone that lacked the SH, G, and F ORFs and that also contained FseI and AscI restriction sites, yielding cDNAs that varied from wild-type (wt) HRSV only in the content of the ORFs at gene positions 6, 7, and 8 (pRS{Delta}SH,G,F and pRS{Delta}SH,F, respectively). Modified areas of the engineered cDNAs were verified by nucleotide sequencing. Infectious viruses were recovered from cDNA as described previously (17), with the following exceptions: (i) in the initial transfection, we included 0.1 µg of a plasmid encoding Gvsv, a chimeric VSIV G protein containing the HRSV F protein cytoplasmic tail domain (17), and (ii) instead of Vero cells, generated viruses were amplified in Vbac cells. At passage 2, recovered virus suspensions were incubated with anti-vaccinia virus Abs (1:50 dilution) and anti-VSIV G Abs (ascitic fluid at a 1:100 dilution) for 1 h at room temperature to neutralize any modified vaccinia Ankara virus that might have carried over independently or via the VSIV G protein transiently expressed in the HEp-2 cells initially transfected. These preincubated virus suspensions were used for the production of passage 3 stocks, which were subsequently titrated by immunoplaque assay as previously described (17), with the following exceptions: (i) Vbac cells were used instead of Vero cells and (ii) to visualize plaques, anti-HRSV nucleocapsid Ab (MAb 6) was used for virus RS{Delta}SH,G,F and anti-HRSV G Ab (L9) was used for virus RS{Delta}SH,F. In addition, for both viruses, plaques were scored based on GFP expression prior to fixation; GFP-expressing plaques overlapped precisely with those identified by immunostaining, as was observed previously (17). Viral RNAs were harvested from cells infected with the engineered virus stocks at passage 3, amplified by reverse transcription-PCR, and verified by nucleotide sequence analysis across cloning junctions and in modified areas. Passage 3 stocks were used for the experiments described below.

Glycoprotein expression. Protein expression by the engineered viruses was examined by cell enzyme-linked immunosorbent assay (CELISA) as previously described (15, 18). Briefly, Vero or Vbac cells infected with the engineered viruses at a multiplicity of 2 were fixed with 4% PF for 15 min at 28 h postinfection and permeabilized by incubation with 0.1% sodium dodecyl sulfate for 5 min at room temperature. Cells were then incubated with Abs against GP64 (AcV5), HRSV G (L9), HRSV F (MAb 19), or HRSV N protein (MAb 6) as a control; washed; and incubated with a horseradish peroxidase-conjugated secondary goat anti-mouse Ab (Pierce). Next, cells were washed and incubated in 1 ml of 3,3',5,5'-tetramethylbenzidine substrate (Pierce). At various times after the addition of the substrate, 100-µl aliquots were collected and added to 2 M sulfuric acid in a 96-well plate to stop the reaction, and the optical density at 450 nm was determined in an ELISA plate reader.

Virus transmission in cell culture. Vero or Vbac cells were plated in six-well plates at approximately 60% confluence, infected at a multiplicity of 0.05 for 1.5 h, and then washed and incubated at 33°C. On days 1, 2, 4, 6, and 8 postinfection (data for day 8 are not shown), one well for each condition was fixed in 4% PF for 15 min and examined for GFP expression on an Olympus IX70 inverted fluorescence microscope. As determined previously, GFP expression provided an accurate indicator of virus infectivity that correlated with PFU (17). Fixed cell cultures were photographed at x200 magnification.

Growth curves. Vero, Vbac, and HEp-2 cells were infected in six-well plates at a multiplicity of 0.25 for 1.5 h at 33°C, washed twice, and then incubated in 1.5 ml of normal growth medium at 33°C. At days 0, 2, and 4 postinfection, the supernatants of infected cells were drawn off, and an equal volume of fresh growth medium was added to the cells. Cells were then scraped into the medium, followed by gentle pipetting. The titers of both the supernatant and cell-derived virus suspensions in Vbac or Vero cells were determined by a 50% tissue culture infective dose (TCID50) assay based on GFP expression from the viral genome, as previously described (17, 18). To determine the total virus titer, supernatant and cell-derived titers were combined. To compare the infectivities of the above-described harvested virus samples to Vero and Vbac cells, frozen virus suspensions from days 0, 2, and 4 postinfection were titrated by the TCID50 assay as described above or were examined by flow cytometry for their ability to enter cells. For the latter procedure, samples were diluted 10 times in normal growth medium and adsorbed to Vero or Vbac cells by using conditions identical to those used for the growth curves (1.5 h at 33°C). At 20 h postinfection, cells were trypsinized, fixed in 4% PF, and analyzed in a FACSCalibur flow cytometer (Becton Dickinson), using 50,000 events per sample.


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RESULTS
 
Generation of a cell line expressing the baculovirus GP64 protein. In order to recover an HRSV in which the SH, G, and F glycoprotein genes were deleted and not functionally replaced, a cell line which could provide a heterologous replacement protein for complementation in trans was generated. As a replacement protein, we used a chimeric form of the baculovirus envelope protein GP64, consisting of the ecto- and transmembrane domains of GP64 coupled to the 12 C-terminal amino acids of the HRSV F protein (GP64/F) (Fig. 1A). GP64/F was previously shown to be expressed and functional in Vero and HEp-2 cells, and cells expressing GP64/F showed no signs of cytopathic effect (18). Moreover, the infectivity of an SH/G/F-depleted HRSV containing GP64/F expressed from the viral genome was substantially more stable than a wt HRSV.



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  		FIG. 1. Characterization of Vbac, a cell line constitutively expressing the GP64/F protein. (A) The GP64/F protein consists of the ecto- and transmembrane domains of the baculovirus GP64 protein coupled to the 12 C-terminal amino acids of the HRSV F protein. TM, transmembrane domain. (B) Analysis of GP64/F surface expression by indirect IF. Vbac cells (passage 8) or Vero cells were incubated on ice with anti-GP64 MAb AcV1, washed, and fixed with 4% PF. Cells were then incubated with an Alexa-594-conjugated secondary Ab (red), stained with Hoechst reagent to visualize nuclei (blue), and examined with fluorescence microscopy (magnification, x400). (C) Flow cytometry analysis. Vbac cells (passage 8) and Vero cells were detached from plates with EDTA, fixed, and incubated with anti-GP64 Abs as described above. Next, cells were incubated with an Alexa-488-conjugated secondary Ab and examined by flow cytometry.

A plasmid constructed to express both GP64/F and a neomycin resistance gene was used to transfect early-passage Vero cells. After incubation at 33°C in G418-containing medium, resistant colonies were selected, expanded, and tested for expression of GP64/F by IF with anti-GP64 MAb AcV5. Cells from colonies expressing the highest levels of GP64/F were then subjected to two rounds of subcloning via limiting dilution and screening by indirect IF. Finally, one cell line (clone 116-25-1) constitutively expressing GP64/F was selected and designated Vbac, passage 1; these cells were slightly larger and had a slightly lower cell division rate than the Vero cells from which they were derived. Figure 1B and C show the expression of GP64/F at the surfaces of Vbac cells at passage 8 compared to that of the parental Vero cell line. GP64/F expression was detected by incubation of cells at 4°C with anti-GP64 MAb AcV1, followed by fixation with 4% PF. After fixation, cells were incubated with an Alexa-594-conjugated secondary Ab, stained with Hoechst reagent (to visualize nuclei), and photographed on a fluorescence microscope (Fig. 1B). Vbac or Vero cells were also detached from plates with 5 mM EDTA, fixed and labeled as described above, and examined for surface GP64/F expression by flow cytometry (Fig. 1C). The data of Fig. 1B and C show that most cells within the Vbac population expressed the GP64/F protein at the cell surface. The expressed protein appeared evenly distributed across the plasma membrane, but expression levels among cells varied (Fig. 1C). The long-term stability of GP64/F expression in the absence of G418 was not investigated. However, when Vbac cells were maintained in medium containing G418, the level and distribution of GP64/F expression remained constant for at least 10 passages.

Construction of cDNAs and recovery of engineered viruses. To generate HRSVs with a modified glycoprotein gene content, two cDNAs were constructed from the A2 strain of HRSV by using methods previously described (17). In one cDNA, the SH, G, and F ORFs were deleted and replaced with the ORFs of three marker proteins, GFP, CAT, and GUS, respectively (pRS{Delta}SH,G,F) (Fig. 2A). This was done to keep the number of genes as in wt HRSV in order to maintain authentic transcription levels. This HRSV cDNA thus encoded no viral envelope proteins. In the second cDNA, the SH and F ORFs were replaced with GFP and GUS, respectively, but the HRSV G protein ORF remained in its native position (pRS{Delta}SH,F). Though altered in the coding potential of genome positions 6, 7, and 8, both cDNAs generated retained fully wt intergenic junctions throughout. Infectious virus was recovered from cDNAs as described previously (Fig. 2B) (17). Briefly, the engineered cDNAs were transfected into HEp-2 cells already infected with modified vaccinia virus Ankara-T7 (30) along with plasmids encoding the nucleocapsid protein (N), phosphoprotein (P), polymerase (L), and transcription factor M2-1 (8). To ensure that the resulting viruses could be passaged from the infected/transfected HEp-2 cells, we included an additional plasmid encoding Gvsv, a chimeric VSIV G protein from which the cytoplasmic tail domain was exchanged with that of the HRSV F protein (17). Supernatants from infected/ transfected HEp-2 cells were transferred onto Vbac cells, and GFP-expressing spreading foci were observed upon incubation at 33°C. The viruses harvested from the first round of Vbac cells, designated RS{Delta}SH,G,F and RS{Delta}SH,F (Fig. 2), were further amplified by one additional passage in Vbac cells, as described in Materials and Methods. Final virus stocks (passage 3) were harvested and examined by reverse transcription-PCR across the modified areas, followed by sequence analysis (data not shown). In Vbac cells, both viruses yielded stock titers of approximately 2 x 106 PFU/ml, approaching titers generally reported for wt HRSV.



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  		FIG. 2. Composition of engineered HRSVs and recovery protocol. (A) From a cDNA of the wt HRSV A2 strain, two cDNAs were constructed with an altered glycoprotein gene content. In construct RS{Delta}SH,G,F, all three HRSV transmembrane glycoprotein ORFs (SH, G, and F) were deleted and replaced with those of GFP, CAT, and GUS, respectively. Construct RS{Delta}SH,F is similar to RS{Delta}SH,G,F; however, the HRSV G protein ORF remained in its native position. (B) Infectious HRSVs were recovered from these cDNAs as previously described (17) with the following modifications: a plasmid encoding a chimeric VSIV G protein, Gvsv (17), was included in the transfection of HEp-2 cells, and amplification of recovered viruses was done in Vbac instead of Vero cells. Passage 2 viruses were incubated with anti-vaccinia ({alpha}-Vacc) and anti-VSIV G ({alpha}-VSIV G) virus Abs as described in Materials and Methods and then used for the generation of passage 3 virus stocks. Passage 3 (pass 3) stocks were titrated in Vbac cells, yielding titers of 2 x 106 PFU/ml.

Protein expression by the engineered HRSVs. To examine the glycoprotein expression by each of the engineered viruses, infected Vero cells were incubated at 33°C and then fixed and permeabilized at 28 h postinfection. Fixed cells were incubated with Abs against GP64 (AcV5), HRSV G (L9), HRSV F (MAb 19), or HRSV N protein (MAb 6) as a control, and protein expression was measured by CELISA (Fig. 3) as previously described (17). A previously described virus, RS{Delta}SH,G,F/GP64/F, was included as a control (18). RS{Delta}SH,G,F/GP64/F lacks the SH, G, and F ORFs, but in contrast to virus RS{Delta}SH,G,F generated in this study, it contains the GP64/F ORF within its genome at position 7 (the ORF order at positions 6, 7, and 8 is GFP-GP64/F-GUS). As a positive control for F expression, a previously engineered virus that expresses the HRSV F protein was included (data not shown). Both of the newly constructed viruses, RS{Delta}SH,G,F and RS{Delta}SH,F, were able to infect Vero cells, as evidenced by the expression of GFP from the viral genomes (data not shown) and by the expression of the HRSV N protein (Fig. 3). In addition to the N protein, virus RS{Delta}SH,F expressed the HRSV G protein but not F or GP64; virus RS{Delta}SH,G,F did not express any transmembrane glycoproteins, as expected. In Vbac cells, both uninfected and infected, the GP64/F protein was detected at a level similar to that seen in Vero cells infected with control virus RS{Delta}SH,G,F/GP64/F. These results show that the glycoprotein expression by the engineered viruses was in agreement with the genome content shown in Fig. 2A and that high levels of GP64/F were present in the Vbac cell line even after infection with the engineered HRSVs.



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  		FIG. 3. Transmembrane glycoprotein expression by HRSVs lacking the SH, G, and F or SH and F ORFs. Protein expression in Vero and Vbac cells infected with the engineered viruses was examined at 28 h postinfection with CELISA as previously described (18). As a comparison, Vero cells infected with virus RS{Delta}SH,G,F/GP64/F were included. RS{Delta}SH,G,F/GP64/F contains the GP64/F gene within its genome as described previously (18). Bars represent the average values from duplicate samples. {alpha}-N, anti-HRSV N protein Ab; {alpha}-G, anti-HRSV G protein Ab; {alpha}-F, anti-HRSV F protein Ab; {alpha}-GP64, anti-GP64 protein Ab; OD450, optical density at 450 nm.

Virus transmission in cell culture. Having a system in which infectivities of glycoprotein depleted HRSVs are solely dependent on the GP64/F protein provided by the complementing cell line allowed us for the first time to monitor HRSV transmission in cell culture in the absence of the SH, G, and F proteins (and without the presence of heterologous viral entry and exit proteins). Vero or Vbac cells were plated at ~60% confluence and infected with viruses RS{Delta}SH,G,F and RS{Delta}SH,F at a multiplicity of 0.05. Cells were incubated at 33°C and examined for GFP expression at daily intervals (Fig. 4). GFP expression from the same position within the genome of an infectious engineered HRSV was previously shown to be an accurate indicator of infectivity that correlated with PFU (17, 18). To examine the spread to neighboring cells, the infected cultures were photographed using fluorescence microscopy on days 1, 2, 4, and 6 postinfection (Fig. 4). As a control, Vero cells infected with a virus containing the GP64/F ORF within its genome (RS{Delta}SH,G,F/GP64/F) were also examined. Viruses RS{Delta}SH,G,F and RS{Delta}SH,F spread rapidly in Vbac cells; by day 6, all cells in the cultures displayed green fluorescence. The rate of spread was similar to that of control virus RS{Delta}SH,G,F/GP64/F in Vero cells (Fig. 4, bottom panels). In contrast, no significant spread to neighboring cells was detected in Vero cell cultures infected with virus RS{Delta}SH,G,F. Although a moderate increase was observed in the number of green cells between days 1 and 2, no increase in the number of cells displaying green fluorescence was observed after day 2. The intensity of fluorescence in infected Vero cells increased over time, resulting by day 4 in single cells that fluoresced very brightly compared to infected Vbac cells. Similar to the results for virus RS{Delta}SH,G,F, a virus expressing the HRSV G protein as its only transmembrane glycoprotein (RS{Delta}SH,F) was also unable to spread to neighboring cells in Vero cultures. Thus, the G protein alone was not sufficient to allow cell-to-cell transmission in Vero cultures, nor did it appear to alter the rate of virus transmission in Vbac cells compared to that of virus RS{Delta}SH,G,F.



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  		FIG. 4. Virus transmission in cell culture. Vero and Vbac cells were plated at ~60% confluence, infected with viruses RS{Delta}SH,G,F and RS{Delta}SH,F at a multiplicity of 0.05, and incubated at 33°C. At days 1, 2, 4, and 6 postinfection, cells were fixed with 4% PF and examined for GFP expression with fluorescence microscopy (magnification, x200). As a comparison, Vero cells infected with the previously described virus RS{Delta}SH,G,F/GP64/F (18) were included.

These results demonstrate that, in contrast to the SH and G proteins, the F protein is critical for HRSV transmission in Vero cell cultures and that expression of the G protein alone does not rescue the transmission deficiency. The reason for the small increase in the number of fluorescing Vero cells between days 1 and 2 has not been investigated, but it may be caused by the fact that GFP expression at day 1 was not sufficiently strong to be detected in all infected cells. Alternatively, a limited amount of virus spread may have occurred through cell division, as cells were plated at 60% confluence and HRSV does not shut down host cell metabolism.

Production of infectious progeny virus. The above data, showing both the expression of GFP and an inability to spread in Vero cells, suggested that viruses RS{Delta}SH,G,F and RS{Delta}SH,F entered Vero cells and replicated their genomes but did not produce infectious virions. To examine infectious virion production, the yields of infectious virus produced in Vbac, Vero, and HEp-2 cells were compared (Fig. 5). Cells were infected with viruses RS{Delta}SH,G,F, RS{Delta}SH,F, and RS{Delta}SH,G,F/GP64/F at a multiplicity of 0.25, washed two times, and then incubated at 33°C. Immediately after infection (day 0), or at 2 and 4 days postinfection, supernatants and cells were harvested separately and titrated on Vbac cells by the TCID50 assay based on the expression of GFP from the viral genome as described previously (17). Supernatant and cell-associated viral titers were combined to determine the total virus yield. In Vbac cells, viruses RS{Delta}SH,G,F and RS{Delta}SH,F replicated to titers similar to that of the control virus RS{Delta}SH,G,F/GP64/F (Fig. 5A), which contains the GP64/F gene within its genome and has previously been shown to yield higher titers than a virus containing the wt G and F proteins in Vero cell cultures (18). A comparison of supernatant and cell-associated titers revealed that, for each virus, the majority of infectivity was associated with the cell fraction, similar to what occurs with wt HRSV (data not shown). In contrast to Vbac cells, when viruses RS{Delta}SH,F and RS{Delta}SH,G,F were used to infect Vero and HEp-2 cells, the yield of infectious virus was approximately 2 orders of magnitude lower than in the Vbac cells. This reduction in infectious virus yield was consistent with the results of Fig. 4, confirming the importance of the F protein for virus infectivity. However, even in the absence of viral transmembrane glycoproteins, there was an approximate 10-fold increase in titers of viruses RS{Delta}SH,G,F and RS{Delta}SH,F grown in Vero and HEp-2 cells when titrated in Vbac cells. This was unexpected given the transmission deficiency shown in Fig. 4. It was unlikely that GP64/F from input virus was responsible for the observed increase, as no GP64/F could be detected by CELISA in Vero cells infected with viruses RS{Delta}SH,G,F and RS{Delta}SH,F at 28 h postinfection (Fig. 3); moreover, analysis by fluorescence microscopy of infected Vero cells fixed at 26 h postinfection with anti-GP64 Abs confirmed the absence of GP64, as shown by CELISA (data not shown).



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  		FIG. 5. Growth curve analysis. Vbac, Vero, and HEp-2 cells were infected with virus RS{Delta}SH,G,F, RS{Delta}SH,F, or RS{Delta}SH,G,F/GP64/F at a multiplicity of 0.25 and incubated at 33°C. Immediately after viral adsorption (day 0) and at days 2 and 4 postinfection (pi), supernatants and cells were harvested separately and titrated on Vbac cells by a TCID50 assay based on the expression of GFP, as described previously (17). Supernatant and cell-associated viral titers were combined to determine the total virus yield.

Because different cell types were used for the virus titrations shown in Fig. 4 and 5 (Vero and Vbac, respectively), we investigated whether the low level of infectivity derived from Vero and HEp-2 cells infected with viruses RS{Delta}SH,G,F and RS{Delta}SH,F, seen in Fig. 5, might be an effect specific to assay on Vbac cells. Selected samples used to determine the titrations shown in Fig. 5 were reexamined. If the engineered viruses were able to produce infectious virions in Vero and HEp-2 cells, their infectivity should be measurable in either GP64/F- or non-GP64/F-expressing cells. Therefore, the infectivities of the stored growth curve samples of virus RS{Delta}SH,G,F and RS{Delta}SH,G,F/GP64/F from HEp-2 and Vero cells (Fig. 5) were compared by titration on Vbac cells or Vero cells, using a TCID50 assay (Fig. 6A). In addition to the TCID50 assay, parallel samples of infected cells were examined with flow cytometry for the expression of GFP as a measure of viral entry (Fig. 6B), as described previously (17). The results shown in Fig. 6A confirmed that, when virus RS{Delta}SH,G,F grown in Vero and in HEp-2 cells was titrated on Vbac cells, modest increases in infectivity were observed. In contrast, when it was titrated on Vero cells, RS{Delta}SH,G,F infectivities of both Vero and HEp-2 cells declined immediately after infection, while those of control virus RS{Delta}SH,G,F/GP64/F did not. Flow cytometry analysis yielded similar results (Fig. 6B). In this assay, virus samples parallel to those used to obtain the results shown in Fig. 6A were diluted 10-fold and used to infect Vero or Vbac cells, and the number of GFP-expressing cells was counted. The percentage of RS{Delta}SH,G,F virions that entered Vbac cells was found to increase from 0.01 to 1.54% (grown in Vero cells) or from 0.01 to 0.43% (grown in HEp-2 cells) from day 0 to 4 postinfection, while no entry into Vero cells was observed (Fig. 6B). Combined, these data show that virus RS{Delta}SH,G,F grown in Vero or HEp-2 cells did not produce infectious virions. The data also suggest that Vbac cells were able to bind and internalize virions produced in the absence of viral transmembrane glycoproteins, presumably mediated by the GP64/F protein expressed at the Vbac cell surface.



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  		FIG. 6. Production of infectious virions in Vero and HEp-2 cells and comparison of titers in Vero and Vbac cells. (A) Samples of viruses RS{Delta}SH,G,F and RS{Delta}SH,G,F/GP64/F from Vero and HEp-2 cells from the experiments whose results are shown in Fig. 5A were titrated on Vbac or Vero cells by a TCID50 assay as described previously (17). Values represent the averages from duplicate samples. pi, postinfection. (B) Tenfold dilutions of the samples used for panel A were adsorbed to Vbac or Vero cells, and GFP-expressing cells were counted with flow cytometry at 20 h postinfection as a measure of viral entry, as previously described (17).


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DISCUSSION
 
Recovery of HRSV lacking the SH, G, and F protein ORFs and containing no functional replacements. While many aspects of HRSV biology have been well studied, neither the assembly and morphology of the infectious unit nor the mechanism by which HRSV spreads from cell to cell is well understood. Early work with the Long strain of HRSV showed significant virus transmission in cell cultures in the presence of neutralizing quantities of pooled sera from convalescent-phase patients, suggestive of a mode of cell-to-cell transmission independent of the transmembrane glycoproteins (23). Subsequent work with a helper virus-dependent mini-replicon system, as well as recovery of viruses lacking the SH and G genes, showed that F protein alone was sufficient to allow virus propagation in Vero cells, although virus lacking the G gene was severely restricted in other cell types and animal models (12, 25-27). Such studies pointed to an important role for the F protein in HRSV propagation. We developed a cell line that expresses a heterologous viral entry and exit protein in order to complement recovery of an HRSV lacking the SH, G, and F protein ORFs and containing no functional replacement ORFs, so that propagation of HRSV in the absence of both homologous and heterologous viral transmembrane glycoproteins could be studied. In previous work, Vero cells proved exceptional compared to other cultured cell lines in that they allowed relatively efficient amplification of an HRSV lacking the G gene, while such a virus was severely restricted in other cell types and in vivo (25, 27). In contrast to the efficient propagation of virus lacking the G gene in Vero cells, the results of this study show that the spread of HRSVs lacking all transmembrane glycoprotein ORFs is impaired in Vero as well as in HEp-2 cells. This demonstrates that the F protein is a critical factor in the ability of HRSV to spread within cell cultures. A virus containing the HRSV G gene as its only transmembrane glycoprotein behaved identically to one lacking all three transmembrane glycoproteins, indicating that the G protein alone was not sufficient to allow virus transmission.

GP64/F-complementing cell line as a tool for studies of the HRSV transmembrane glycoproteins. The importance of the F protein is most likely related to its membrane fusion function (28), which allows the virus to cross the plasma membrane to achieve virus entry. In addition, the F protein may provide other functions that are critical for virus propagation, such as functions in assembly, budding, and morphology. A supportive role for the F protein in cell attachment has already been demonstrated (5), and recently a "chaperoning" of F function by the G protein has been suggested. Having demonstrated efficient recovery of HRSVs lacking a functional membrane fusion protein, the Vbac line constitutes a powerful system to study the roles and relative contributions of the transmembrane glycoproteins in the above-mentioned aspects of HRSV biology. Independence from functional SH, G, and F proteins allows for the recovery of HRSVs with any desired glycoprotein content, so that both the roles of individual proteins and the importance of interactions between them can be studied. Importantly, this can be done at wt protein levels due to the replacement of deleted genes with place keeper genes, maintaining authentic transcription and replication levels (17). Moreover, viruses functionally complemented by the GP64/F protein have the potential to be significantly more stable, based on previous work showing enhanced stability at 4°C (18), further facilitating HRSV studies.

Trans-complementation as an alternate approach for vaccine development. To date, it has proven challenging to find an acceptable balance between immunogenicity and the safety of live HRSV vaccine candidates (6, 16). The ability to generate infectious virus by complementation with a heterologous viral protein from a cell line may provide new opportunities to address vaccine safety. With the Vbac cell system, viruses that are impaired in cell-to-cell transmission yet should not be attenuated for replication and thus expression of viral antigens can be generated. Also, trans-complementation removes the dependence on functional G and F protein for virus propagation and stock production, allowing one to include selective G and F protein domains or epitopes (or nonfunctional versions of the F protein) into an engineered genome. Alternatively, when included in the engineered HRSV genome, the GP64/F protein conferred onto the virus a temperature-sensitive phenotype, with reduced propagation at 37°C (18). These possibilities, together with enhanced HRSV stability via the use of the GP64/F protein as shown previously (18), may provide new advantages in our ability to find a balance between safety and efficacy and to improve the stability of HRSV, two key problematic areas in the design of live attenuated vaccines.

Conclusions. A cell line complementation system based on a heterologous viral protein, baculovirus GP64, allowed the recovery of HRSVs that lack any transmembrane glycoprotein gene. Cell culture experiments with the resulting engineered viruses showed that these viruses are competent to infect cultured Vero and HEp-2 cells, express their genes, and replicate but that they cannot spread. Further, these studies provided direct evidence that the F protein is critical for cell-to-cell transmission of HRSV. The efficiency with which glycoprotein gene-deficient HRSVs were recovered demonstrates that the approach of providing viral proteins in trans constitutes a powerful system to study the role of the individual HRSV SH, G, and F proteins, as well as interactions between them, in HRSV biology.


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ACKNOWLEDGMENTS
 
We thank the members of the G. W. Wertz and L. A. Ball laboratories for helpful discussions during the preparation of the manuscript and Gary W. Blissard for providing the GP64 construct and antibodies.

This work was supported by Public Health Service grant AI20181 from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, The Medical School, University of Alabama at Birmingham, BBRB Room 366, 845 19th St. South, Birmingham, AL 35294. Phone: (205) 934-0877. Fax: (205) 934-1636. E-mail: gailw{at}uab.edu. Back


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REFERENCES
 
    1
  1. Blissard, G. W., and J. R. Wenz. 1992. Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J. Virol. 66:6829-6835.[Abstract/Free Full Text]
    2. 2
  2. Buchholz, U. J., S. Finke, and K.-K. Conzelmann. 1999. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J. Virol. 73:251-259.[Abstract/Free Full Text]
    3. 3
  3. Bukreyev, A., S. S. Whitehead, B. R. Murphy, and P. L. Collins. 1997. Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse. J. Virol. 71:8973-8982.[Abstract]
    4. 4
  4. Couch, R. B., J. A. Englund, and E. Whimbey. 1997. Respiratory viral infections in immunocompetent and immunocompromised persons. Am. J. Med. 102(3A):2-9. [Discussion, 102(3A):25-26.][Medline]
    5. 5
  5. Feldman, S. A., S. Audet, and J. A. Beeler. 2000. The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate. J. Virol. 74:6442-6447.[Abstract/Free Full Text]
    6. 6
  6. Graham, B. S., J. A. Rutigliano, and T. R. Johnson. 2002. Respiratory syncytial virus immunobiology and pathogenesis. Virology 297:1-7.[CrossRef][Medline]
    7. 7
  7. Han, L. L., J. P. Alexander, and L. J. Anderson. 1999. Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden. J. Infect. Dis. 179:25-30.[CrossRef][Medline]
    8. 8
  8. Hardy, R. W., S. B. Harmon, and G. W. Wertz. 1999. Diverse gene junctions of respiratory syncytial virus modulate the efficiency of transcription termination and respond differently to M2-mediated antitermination. J. Virol. 73:170-176.[Abstract/Free Full Text]
    9. 9
  9. Hefferon, K. L., A. G. Oomens, S. A. Monsma, C. M. Finnerty, and G. W. Blissard. 1999. Host cell receptor binding by baculovirus GP64 and kinetics of virion entry. Virology 258:455-468.[CrossRef][Medline]
    10. 10
  10. Hendricks, D. A., K. Baradaran, K. McIntosh, and J. L. Patterson. 1987. Appearance of a soluble form of the G protein of respiratory syncytial virus in fluids of infected cells. J. Gen. Virol. 68:1705-1714.[Abstract/Free Full Text]
    11. 11
  11. Huang, Y. T., P. L. Collins, and G. W. Wertz. 1985. Characterization of the 10 proteins of human respiratory syncytial virus: identification of a fourth envelope-associated protein. Virus Res. 2:157-173.[CrossRef][Medline]
    12. 12
  12. Karron, R. A., D. A. Buonagurio, A. F. Georgiu, S. S. Whitehead, J. E. Adamus, M. L. Clements-Mann, D. O. Harris, V. B. Randolph, S. A. Udem, B. R. Murphy, and M. S. Sidhu. 1997. Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc. Natl. Acad. Sci. USA 94:13961-13966.[Abstract/Free Full Text]
    13. 13
  13. Kumar, M., B. P. Bradow, and J. Zimmerberg. 2003. Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64. Hum. Gene Ther. 14:67-77.[CrossRef][Medline]
    14. 14
  14. Levine, S., R. Klaiber-Franco, and P. R. Paradiso. 1987. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J. Gen. Virol. 68:2521-2524.[Abstract/Free Full Text]
    15. 15
  15. Monsma, S. A., and G. W. Blissard. 1995. Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein. J. Virol. 69:2583-2595.[Abstract]
    16. 16
  16. Murphy, B. R., and P. L. Collins. 2002. Live-attenuated virus vaccines for respiratory syncytial and parainfluenza viruses: applications of reverse genetics. J. Clin. Investig. 110:21-27.[CrossRef][Medline]
    17. 17
  17. Oomens, A. G. P., A. G. Megaw, and G. W. Wertz. 2003. Infectivity of a human respiratory syncytial virus lacking the SH, G, and F proteins is efficiently mediated by the vesicular stomatitis virus G protein. J. Virol. 77:3785-3798.[Abstract/Free Full Text]
    18. 18
  18. Oomens, A. G. P., and G. W. Wertz. 2004. The baculovirus GP64 protein mediates highly stable infectivity of a human respiratory syncytial virus lacking its homologous transmembrane glycoproteins. J. Virol. 78:124-135.[Abstract/Free Full Text]
    19. 19
  19. Pastey, M. K., J. E. Crowe, Jr., and B. S. Graham. 1999. RhoA interacts with the fusion glycoprotein of respiratory syncytial virus and facilitates virus-induced syncytium formation. J. Virol. 73:7262-7270.[Abstract/Free Full Text]
    20. 20
  20. Pringle, C. R. 1987. Progress towards control of the acute respiratory viral diseases of childhood. Bull. W. H. O. 65:133-137.[Medline]
    21. 21
  21. Roberts, S. R., D. Lichtenstein, L. A. Ball, and G. W. Wertz. 1994. The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons. J. Virol. 68:4538-4546.[Abstract/Free Full Text]
    22. 22
  22. Schlender, J., G. Zimmer, G. Herrler, and K.-K. Conzelmann. 2003. Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G, determines the specificity of RSV infection. J. Virol. 77:4609-4616.[Abstract/Free Full Text]
    23. 23
  23. Shigeta, S., Y. Hinuma, T. Suto, and N. Ishida. 1968. The cell to cell infection of respiratory syncytial virus in HEp-2 monolayer cultures. J. Gen. Virol. 3:129-131.[Abstract/Free Full Text]
    24. 24
  24. Stope, M. B., A. Karger, U. Schmidt, and U. J. Buchholz. 2001. Chimeric bovine respiratory syncytial virus with attachment and fusion glycoproteins replaced by bovine parainfluenza virus type 3 hemagglutinin-neuraminidase and fusion proteins. J. Virol. 75:9367-9377.[Abstract/Free Full Text]
    25. 25
  25. Techaarpornkul, S., N. Barretto, and M. E. Peeples. 2001. Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J. Virol. 75:6825-6834.[Abstract/Free Full Text]
    26. 26
  26. Teng, M. N., and P. L. Collins. 1998. Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles. J. Virol. 72:5707-5716.[Abstract/Free Full Text]
    27. 27
  27. Teng, M. N., S. S. Whitehead, and P. L. Collins. 2001. Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo. Virology 289:283-296.[CrossRef][Medline]
    28. 28
  28. Walsh, E. E., and J. Hruska. 1983. Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein. J. Virol. 47:171-177.[Abstract/Free Full Text]
    29. 29
  29. Whitehead, S. S., A. Bukreyev, M. N. Teng, C.-Y. Firestone, M. St. Claire, W. R. Elkins, P. L. Collins, and B. R. Murphy. 1999. Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees. J. Virol. 73:3438-3442.[Abstract/Free Full Text]
    30. 30
  30. Wyatt, L. S., B. Moss, and S. Rozenblatt. 1995. Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells. Virology 210:202-205.[CrossRef][Medline]

Journal of Virology, September 2004, p. 9064-9072, Vol. 78, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.17.9064-9072.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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