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Journal of Virology, September 2007, p. 9490-9501, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00364-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice{triangledown}

Christine D. Krempl,1,2* Anna Wnekowicz,1 Elaine W. Lamirande,2 Giw Nayebagha,1 Peter L. Collins,2 and Ursula J. Buchholz2

Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany,1 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-80072

Received 20 February 2007/ Accepted 6 June 2007


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ABSTRACT
 
Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene ({Delta}G) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the {Delta}G mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.


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INTRODUCTION
 
Human and bovine respiratory syncytial viruses (HRSV and BRSV, respectively) are the most extensively characterized representatives of the genus Pneumovirus, subfamily Pneumovirinae, family Paramyxoviridae. HRSV and BRSV are important agents of severe lower respiratory tract infections in their respective hosts. Reverse genetics systems are available for HRSV and BRSV (9, 10) and have been useful for determining the functions of viral proteins and RNA signals on a molecular level. However, in vivo characterization of the contribution of these factors to viral pathogenesis has been hampered by the difficulty of performing defined experiments in the natural host. Mice, cotton rats, and monkeys are frequently used as experimental animal models of HRSV infection, but viral replication is only semipermissive and disease is either lacking or minimal (29). Chimpanzees approach humans with regard to the magnitude of HRSV replication and disease but are inconvenient, expensive, and scarce (45). BRSV has been used as a surrogate model of HRSV infection. The genome sequence is closely related to that of HRSV (9), and infection of calves can result in a disease resembling that caused by HRSV in humans (reviewed in reference 29). However, the virulence of BRSV in experimental infections can be variable, and there are limitations to the manageability of this large-animal model and the availability of immunological tools (38, 43).

Pneumonia virus of mice (PVM), a murine member of the genus Pneumovirus, is another potential surrogate for HRSV (15). PVM was first isolated by Horsfall and Hahn in the 1930s when mice were used during an attempt to isolate new human respiratory viruses (21, 22). Mice were inoculated with nasopharyngeal aspirates from patients with acute noninfluenza respiratory tract disease, followed by serial passage of mouse lung homogenates from animal to animal. Following several passages, the mice frequently developed morbidity and fatal pneumonia. PVM was isolated and identified as a potential murine pathogen rather than a human pathogen. Thus, the natural history of PVM is unclear, and reports of naturally occurring infection and disease are rare and limited to immunocompromised animals (36, 44). Infection of immunocompetent animals is thought to be inapparent or latent. However, PVM infections of other rodents have been described, and antibodies specific to PVM or a serologically related virus have been detected in many rodent species as well as in other mammals, including humans (16, 20, 28, 31, 33).

Two laboratory strains, strain 15 and strain J3666, have been described for PVM. An isolate of strain 15 has been used as a prototype in several experiments (13, 34), although this now appears to be a nonpathogenic mutant that was attenuated by passage in primate cell culture. However, we recently showed that the isolate of strain 15 that is available from the American Type Culture Collection (ATCC) is a virulent virus that thus resembles the original strain 15 isolate described by Horsfall and Hahn (21-23). The second laboratory strain of PVM, strain J3666, is virulent in mice and has been maintained entirely by passage in mice (12, 34). Presumably it was isolated at approximately the same time and place as strain 15 (42). However, there are no reports of the isolation in the literature, and thus, its origin is somewhat unclear. Strain J3666 has been used for in vivo studies of pathogenicity.

Recently, complete genomic sequences were reported for the virulent strain 15, its nonpathogenic variant, and strain J3666 (24, 42). The prototypic virulent PVM strain 15 has a nonsegmented negative-sense RNA genome of 14,886 nucleotides (nt) (24). The genome contains 10 genes that encode 12 proteins. Eleven PVM proteins appear to correspond to the following HRSV proteins: the nucleocapsid protein N; the phosphoprotein P; the large polymerase protein L; the M2-1 and M2-2 proteins, which are encoded by overlapping open reading frames (ORFs) in the M2 mRNA and are involved in RNA synthesis (3, 11, 18); the matrix protein M; the nonstructural NS1 and NS2 proteins, involved in inhibiting the host interferon system (7, 27, 37, 39, 43); and three transmembrane surface glycoproteins, namely, the G protein, which is involved in attachment, the F protein, which is involved in membrane fusion, and the small hydrophobic protein, which might be an antiapoptotic factor (46). The 12th PVM protein does not have a counterpart in HRSV or BRSV; it is a 137-amino-acid protein of unknown function that is encoded by a second ORF in the P mRNA (2).

Studies of PVM pathogenesis to date have compared strain J3666 as the virulent strain with the nonpathogenic version of strain 15 as the nonpathogenic strain (see, e.g., references 13, 34, and 42). The recent sequence analysis mentioned above showed that these two viruses differ by 59 nucleotide substitutions involving 37 amino acid substitutions, complicating the identification of the relevant differences. In addition, the G gene of the nonpathogenic variant of PVM strain 15 appears to have sustained a single-nucleotide insertion in a run of four U residues at gene positions 169 to 172 (GenBank accession number AY729016), which immediately follows codon 29 in the G ORF. This would shift translation into a second frame that terminates after six additional codons. However, 5 nt upstream of this termination site, there is a second AUG in the G ORF (codon 34), and initiation there would yield a G protein that lacks the first 33 amino acids at its N terminus. The pneumovirus G protein is a type II glycoprotein: in the case of PVM, amino acids 1 to 34 are predicted to constitute its cytoplasmic tail, amino acids 35 to 59 would constitute its signal/anchor sequence, and the remainder of the molecule would be the ectodomain. Thus, initiation at Met-34 would eliminate essentially the entire cytoplasmic tail. This mutation was yet another candidate to be involved in the attenuation phenotype of the nonpathogenic version of strain 15, but it was necessary to evaluate this in a defined genetic background.

In the present study, we developed a reverse genetic system for the virulent PVM strain 15 and used this system to study the contribution of the G protein, and in particular its cytoplasmic tail, to virulence. We constructed two G mutants that were found to be highly attenuated, but for strikingly different reasons. Specifically, a virus lacking the complete G protein was nonpathogenic due to drastic decreases in replication and viral load in vivo. In contrast, a virus lacking only the cytoplasmic tail of G was nonpathogenic despite a level of in vivo replication indistinguishable from that of its wild-type parent. These mutants demonstrate a major role of the cytoplasmic tail of G in pathogenicity in vivo and illustrate the utility of this reverse genetic system for the study of pneumovirus virulence factors in a susceptible in vivo model.


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MATERIALS AND METHODS
 
Viruses and cells. PVM virulent strain 15 and its recombinant derivatives were propagated in BHK-21 (ATCC CCL-10) cells as described previously (24). BHK-21 cells and the BHK-derived BSR-T7/5 cells (9) were maintained in Glasgow minimal essential medium (MEM) (Invitrogen GIBCO), and Vero cells (ATCC CCL-81) were maintained in Eagle's MEM (Biochrom), all supplemented with 10% fetal calf serum. Virus titers were determined by a plaque assay on Vero cells under 0.8% methylcellulose for 5 days at 32°C, followed by fixation with 80% methanol. Plaques were visualized by incubation with a polyclonal rabbit serum raised against the PVM G protein, followed by incubation with biotinylated goat anti-rabbit immunoglobulin G (IgG) (Pierce) as a second antibody. Bound antibodies were detected by incubation with streptavidin coupled to horseradish peroxidase (HRP) (BD Pharmingen), followed by color development by incubation with 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate (Merck). Growth curves were performed in replicate wells. At the indicated time points, duplicate wells were harvested by scraping the cells into the overlying medium. The cells were pelleted by low-speed centrifugation, subjected to three cycles of freeze-thawing in a small volume of medium, and centrifuged a second time. The supernatant was harvested, combined with that from the first centrifugation, flash frozen, and stored for titration. Each aliquot was titrated in duplicate, and the mean was calculated.

RNA isolation, RT-PCR, and nucleotide sequencing. Total cellular RNA or virion-associated RNA (vRNA) was isolated as described previously (24). For reverse transcription (RT), RNA was mixed with 5 pmol of a specific primer, incubated for 10 min at 70°C, and assembled for an RT reaction that was performed using Superscript II reverse transcriptase (Invitrogen) at 50°C for 1 h, followed by digestion of DNA-RNA hybrids with RNase H at 37°C for 20 min. PCR was performed with Pfx polymerase (Invitrogen) at 68°C. Nucleotide sequence analysis was performed using an ABI 3100 sequencer with the BigDye Terminator ready reaction kit, version 1.1 (Applied Biosystems).

Construction of PVM cDNAs. All cDNAs are based on the consensus sequence of the virulent PVM strain 15 (GenBank accession number AY729016) (24). For construction of support plasmids expressing the nucleoprotein N, the phosphoprotein P, and the putative transcription factor M2-1, the respective ORFs were amplified from vRNA by RT-PCR using specific primers and were cloned into the NcoI restriction site of the T7 expression vector pTM1 such that the ATG of the NcoI site became the translational start site of the ORF (17). The expression plasmid carrying the ORF of the RNA polymerase was assembled from two overlapping cDNA fragments derived by RT-PCR such that it could also be used as a shuttle vector for construction of the full-length plasmid (Fig. 1A). Fragment 1, of 2,895 nt (fragment 4 of the full-length construct), encompassed a naturally occurring BamHI site, which contained the last nucleotide of the translation initiation codon of the L ORF, and an XhoI site (nt 8475 to 8,480 and 11374 to 11,379, respectively, of PVM 15). This fragment was cloned into the BamHI-XhoI window of pT7 HMPV L, a plasmid encoding the L protein of human metapneumovirus (HMPV) (5), thereby replacing the HMPV sequence. Fragment 2, of 3,541 nt (fragment 5 of the full-length construct), encompassed the residual part of the L gene including the XhoI site and the L gene end (GE), as well as the complete PVM 15 trailer sequence and 35 nt of the sequence of the hepatitis D virus (HDV) ribozyme. This fragment was cloned into a separate pTM1 vector. After confirmation of the sequence, the second L fragment was transferred into the XhoI-StuI window of pTM pvmL1, thus creating pTM pvmL.


Figure 1
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  		FIG. 1. Construction of plasmids expressing the antigenomes of rPVM and rPVM-GFP7. (A) Map of the antigenome of rPVM showing the locations of the viral genes and, below, the five overlapping cDNA fragments (numbered 1 to 5) used to construct its cDNA. PT7, T7 RNA polymerase promoter; {delta}, hepatitis delta virus ribozyme; TT7, T7 RNA polymerase terminator. Unique restriction enzyme sites used to construct the cDNA are given, with their nucleotide positions in parentheses; the AgeI and BstBI sites that were created during construction are underlined. (B) Nucleotide changes introduced into rPVM to create AgeI (upper panel) and BstBI (lower panel) markers in the SH-G and M2-L intergenic regions, respectively. Predicted transcription GS and GE signals are boldfaced, intergenic sequences are roman, restriction enzyme sites are underlined, and nucleotide substitutions and additions in rPMV are italicized, while deletions are indicated by gaps. (C) Insertion of a transcription cassette encoding enhanced GFP into the AgeI site of rPVM, placing GFP as the seventh gene in the 3'-to-5' order. The GFP ORF (open rectangle, not drawn to scale) was modified by PCR to add a GS sequence derived from the N gene (which also is identical in the P gene) on the upstream side of the ORF and a GE sequence derived from the F gene on the downstream side (each underlined). The fragment was flanked by AgeI restriction sites whose positions in the final cDNA are indicated.

For construction of the plasmid encoding the complete antigenome of PVM 15 (Fig. 1), plasmid pKS II 3/7 HDV-T7ter, containing the HDV ribozyme followed by a terminator for the T7 RNA polymerase (14), was modified to contain a polylinker with NheI, AgeI, and BstBI sites, as well as the first 17 nt of the PVM L gene, including the gene start (GS) signal and the naturally occurring BamHI site. This polylinker was cloned into the XmaI-RsrII window of p3/7 HDV-T7ter to accept the BamHI-RsrII fragment of pTM pvmL and three overlapping subgenomic cDNA fragments, described below, that were generated by RT-PCR from vRNA. Fragment 1 (2,727 nt) comprised the promoter for the T7 RNA polymerase, three G residues to enhance promoter activity, the PVM leader, NS1, NS2, N, and 441 nt of the P gene, flanked on the upstream end by an XmaI site and on the downstream end by a naturally occurring NheI site at position 2702 (relative to the PVM genome). Fragment 2 (1,807 nt) overlapped with the NheI site on the upstream end and contained the residual 472 nt of the P gene and the complete M and SH genes. An AgeI marker restriction site that would be located in the intergenic region between the SH and G genes in the final plasmid was added on the downstream site of fragment 2 (Fig. 1B). Fragment 3 encompassed the complete G, F, and M2 genes. On the upstream end, the fragment overlapped with the added AgeI site, whereas a BstBI restriction site that would be located in the M2-L intergenic region was added to the downstream end. The subgenomic fragments were cloned and the sequences confirmed. Confirmed fragments were added to the corresponding restriction sites of the vector described above containing the PVM L gene, thus creating pPVM. The sequence of the complete assembled antigenome was confirmed in its entirety.

To construct a variant encoding green fluorescent protein (GFP), pPVM-GFP7, the pPVM plasmid was modified by addition of a transcription cassette containing the ORF for enhanced GFP (Clontech, Inc.) to the AgeI restriction site in the SH-G intergenic region (Fig. 1C). To create the GFP insertion cassette, the GFP ORF was amplified by PCR to add an AgeI site, followed by the GS sequence of the PVM N gene (which is identical to that of the P gene), and then the Kozak sequence, on the upstream side and the GE sequence of the PVM F gene, followed by an AgeI site, on the downstream end (Fig. 1C). The structure of the GFP transcription cassette was confirmed by sequencing.

To construct a mutant in which the G gene was deleted (pPVM-GFP-{Delta}G), the F-M2 portion of pPVM-GFP7 was amplified with a forward primer that added an AgeI site at the upstream side of the F GS and the same reverse primer that was used to add the BstBI site to the downstream end of the M2 gene, as shown with fragment 3 of pPVM1 (Fig. 1A). The PCR product was digested with AgeI and BstBI and then cloned into the AgeI-BstBI window of pPVM-GFP7. This in effect replaced the AgeI-BstBI fragment containing the G, F, and M2 genes (Fig. 1A) with an AgeI-BstBI fragment containing only the F and M2 genes, thus deleting the G gene.

To construct a mutant in which the cytoplasmic tail of G was deleted (pPVM-GFP-Gt), the pPVM-GFP7 cDNA was subjected to PCR using a forward primer that contained the AgeI restriction site and G GS sequence, followed by 22 nt of the G ORF starting at nt 178 of the G gene. PCR amplification using this forward primer and the same reverse primer as for the generation of fragment 3 resulted in a cDNA containing a truncated G gene with the GS sequence fused to nt 178 of the G gene (thereby deleting the intervening167 nt), the complete naturally occurring G gene downstream of nt 178, and the G-F intergenic region, followed by the complete downstream part of fragment 3 of pPVM1. The insert was then digested with AgeI and BstBI and cloned into the corresponding window of pPVM, resulting in pPVM-Gt. The antigenomic cDNAs of pPVM-{Delta}G and pPVM-Gt were further modified by insertion of a GFP transcription cassette into the AgeI site as described for the generation of pPVM GFP7, thereby generating pPVM-GFP-{Delta}G and pPVM-GFP-Gt, respectively. All regions of the final full-length cDNA clones that had been amplified by PCR were confirmed completely by sequence analysis.

Recovery of recombinant viruses. Plasmids were transfected into BSR T7/5 cells as described previously for recovery of recombinant BRSV (rBRSV) and rHMPV (5, 9), with minor modifications to adjust for optimal growth conditions for PVM. Briefly, BSR T7/5 cells in 35-mm-diameter wells were transfected with 5 µg of plasmid pPVM or a derivative, 2 µg each of pTM pvmN and pTM pvmP, and 1 µg each of pTM pvmM2-1 and pTM pvmL using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After incubation at 37°C overnight, the transfection medium was replaced with Glasgow MEM supplemented with 5% fetal calf serum, and the culture was maintained at 32°C. Four days after transfection, cells were trypsinized, cocultivated with an equal number of BHK-21 cells, and incubated for as long as 6 days to yield passage 1 virus.

Analysis of secreted proteins. To isolate soluble proteins, two-thirds of the medium overlying an infected-cell monolayer was harvested and subjected to low-speed centrifugation to remove cell debris, followed by ultracentrifugation at 100,000 x g for 90 min to remove virus particles. Soluble proteins contained in the resulting supernatant were concentrated 50-fold using an Amicon centrifugal filter device (Millipore). After the addition of an equal volume of 2x sodium dodecyl sulfate (SDS)/Tris buffer (nonreducing), the samples were heat denatured, subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (10% gel), and transferred to supported nitrocellulose membranes.

To purify virus particles, the infected cells from the same culture were scraped into the remaining one-third of the overlying medium and processed by freeze-thawing as described under "Viruses and cells" above. Virus particles were pelleted from the supernatant by ultracentrifugation through a 30% sucrose cushion (at 100,000 x g for 90 min). Following lysis with 1x SDS/Tris buffer (nonreducing), the samples were heat denatured and subjected to SDS-PAGE as described above.

The membranes were incubated with one of the following antibody preparations for detection of viral proteins: biotinylated polyclonal HRSV-specific goat antibodies (Biogenesis, Poole, United Kingdom), followed by detection with streptavidin-conjugated HRP; rabbit serum raised against the PVM G protein, followed by detection with biotinylated goat anti-rabbit IgG (Pierce) and streptavidin-conjugated HRP; or serum from a mouse that had recovered from infection with PVM, followed by detection with HRP-conjugated rabbit anti-mouse IgG (DAKO). Bound antibodies were visualized and documented by the chemiluminescence detection system ChemiDoc XRS (Bio-Rad).

Mouse studies. For infection with PVM, BALB/c mice were anesthetized with a mixture of ketamine and xylazine (100 mg and 5 mg per kg of body weight, respectively, given intraperitoneally) and inoculated intranasally with the indicated viruses diluted with phosphate-buffered saline to a final volume of 80 µl. For determination of virulence, mice were weighed daily and monitored by visual inspection twice per day. The main visual disease signs were ruffled fur, hunching, and listless behavior. Mice were sacrificed by cervical dislocation if the weight loss exceeded 25% of the weight on day 0 or if the animals were obviously in extremis. To determine the extent of virus replication, mice were sacrificed on the indicated day, and lungs and nasal turbinates were removed separately for virus quantification by a plaque assay. All experiments involving animals were approved by the animal ethics committee of the Regierungspräsidium Freiburg.


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RESULTS
 
Reverse genetic system for virulent PVM strain 15. A plasmid encoding the complete antigenomic RNA of virulent PVM strain 15 (pPVM) was constructed from five overlapping subgenomic RT-PCR fragments as illustrated in Fig. 1A. As markers, a unique AgeI site was added to the SH-G intergenic region by the substitution of 1 and addition of 3 nt, increasing its length from 2 nt to 5 nt, and a unique BstBI site was added to the M2-L intergenic region by the addition of 2 and deletion of 1 nt, increasing its length from 9 nt to 10 nt. The encoded rPVM antigenome would be 14,890 nt long, compared to 14,886 nt for its biological parent. Apart from these markers, the sequence of the rPVM cDNA was identical to the consensus sequence of its biological parent (GenBank accession number AY729016) (24).

To facilitate virus detection, we also made a version of pPVM containing a gene encoding GFP. PCR was used to construct a transcription cassette containing the ORF of enhanced GFP under the control of the GS signal of N (which also is identical to that of the P gene) and the GE signal of F, flanked by AgeI sites (Fig. 1C). This cassette was inserted in the AgeI site of pPVM. Thus, the resulting plasmid, pPVM-GFP7, contained 11 genes, with the added GFP gene located at position 7 between the SH and G genes. The insert increased the genome length to 15,642 nt.

Recovery of recombinant PVM. Plasmid pPVM or pPVM-GFP7 was transfected, together with T7 expression plasmids encoding the N, P, M2-1, and L support proteins, into BSR-T7/5 cells that constitutively express T7 RNA polymerase (5, 9). In cultures transfected with pPVM-GFP7, individual GFP-expressing cells were detected 2 days after transfection. During the following days, foci of infected cells formed, followed by spreading over the residual monolayer. Final titers of both recombinant viruses reached more than 107 PFU/ml after the second passage. To confirm the recombinant origin of the recovered viruses, total intracellular RNA was isolated from infected BHK-21 cells and RT-PCR was used to amplify a fragment spanning nt 3312 (in the N gene) to 5981 (in the F gene) (positions relative to PVM15), which included the AgeI marker restriction site and the added GFP gene. Detection of the predicted PCR products depended on the presence of reverse transcriptase, indicating that the products represented RNA (data not shown). Restriction digestion confirmed the presence of the AgeI site in rPVM and the presence of the GFP insert in rPVM-GFP7, whereas neither was present in the biologically derived parent (Fig. 2A).


Figure 2
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  		FIG. 2. In vitro analysis of recovered rPVM and rPVM-GFP7. (A) RT-PCR and restriction analysis of viral RNA from BHK-21 cells infected with the biological wild-type virus PVM15 (lanes 1 and 2), the wild-type virus rPVM (lanes 3 and 4), or rPVM-GFP7 (lanes 5 and 6). The RT-PCR products were made using primers that hybridized within the N and F genes, spanning PVM positions 3312 to 5981. The amplified fragments were digested with AgeI (lanes marked +) or left untreated (lanes marked –) and electrophoresed on a 1% agarose gel. The predicted sizes of the fragments are given on the left. The last lane represents a 1-kbp DNA ladder. (B) Multistep growth kinetics of biological and recombinant PVMs in vitro. Replicate monolayers of BHK-21 cells were infected with the biological parental virus PVM15, rPVM, or rPVM-GFP7 at an input MOI of 0.01 PFU per cell. At the indicated time points, the cells and medium were harvested, freeze-thawed, clarified, and flash frozen. Virus titers were determined by a plaque assay. Error bars, standard errors of the means. (C) Cells from the experiment for which results are shown in panel B were examined for GFP expression on days 1 to 4 using an inverted fluorescence microscope.

The in vitro replication efficiencies of rPVM and rPVM-GFP7 were compared to that of biologically derived PVM strain 15 (PVM15) in a multicycle time course in BHK-21 cells. Cell monolayers were infected in duplicate at a multiplicity of infection (MOI) of 0.01 PFU/cell, and cells and medium supernatants were harvested at 24-h intervals and stored for subsequent viral titration by a plaque assay as described in Materials and Methods. As shown in Fig. 2B, the kinetics and viral yields of PVM15, rPVM, and rPVM-GFP7 were essentially the same, reaching titers of more than 107 PFU/ml by day 7. Monitoring of the expression of GFP by fluorescent microscopy revealed a continuous spread of rPVM-GFP7 up to day 4, at which time almost 100% of the cells were expressing GFP (Fig. 2C). Interestingly, the monolayer stayed viable up to the end of the experiment at day 7, continuing to produce infectious particles and with no sign of syncytium formation. This was also observed for BHK-21 monolayers infected with PVM15 and rPVM and for Vero cell monolayers infected with each of the three viruses (data not shown).

Replication and virulence of rPVM and rPVM-GFP7 in mice. To investigate the virulence of the recombinant viruses, BALB/c mice were infected intranasally with graded doses of biologically derived PVM15 (500 PFU and 50 PFU), rPVM (5,000 PFU, 500 PFU, and 50 PFU), or rPVM-GFP7 (5,000 PFU and 500 PFU). The mice were observed closely and weighed daily.

All of the mice that received 500 PFU of PVM15 or rPVM died or were sacrificed in extremis by day 8 and day 10, respectively (Fig. 3A). However, the onset of symptomatic disease (day 5 versus day 6) (Fig. 3B) and occurrence of first fatalities (day 7 versus day 8) (Fig. 3A) were delayed 1 to 2 days for rPVM, indicating a modest degree of attenuation of rPVM compared to its biological parent. This attenuation was more pronounced in infections involving 50 PFU. In this case, all of the animals infected with rPVM survived (Fig. 3A), although substantial disease was evident, as indicated by weight loss (Fig. 3B). In contrast, following infection with 50 PFU of the biological wild-type virus, most of the mice had to be sacrificed in extremis, and just one mouse survived. The median lethal dose was calculated to be 40 PFU for PVM15 and 160 PFU for rPVM, indicating a fourfold attenuation for rPVM (35).


Figure 3
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  		FIG. 3. Virulence of wild-type and recombinant PVM. Six- to eight-week-old BALB/c mice were infected intranasally with the indicated doses of PVM15, rPVM, and rPVM-GFP7 in an inoculum volume of 80 µl. Mice were observed closely and weighed daily. Mice that met the end point regulations of the German animal protection board were euthanized by cervical dislocation. The results of two independent experiments, each including at least four animals per group, were combined. (A) Survival after infection with 500 PFU (PVM15, rPVM, and rPVM-GFP7) or 50 PFU (PVM15 and rPVM) of the indicated viruses. The total numbers of animals are given beneath the doses. (B) Body weight (relative to that on day 0, taken as 100%) following infection with the indicated viruses at the indicated doses.

Although the insertion of GFP into the genome of rPVM did not have a detectable effect on replication in vitro, it had a modest attenuating effect in vivo. Fifty percent of the mice infected with 500 PFU of rPVM-GFP7 survived (Fig. 3A), although they experienced substantial weight loss (Fig. 3B). In this comparison, the onset of symptomatic disease, as indicated by ruffled fur and weight loss, was delayed 1 day for rPVM-GFP7 compared to rPVM (Fig. 3B and data not shown). Based on this 50% lethal dose (LD50) of 500 PFU, rPVM-GFP7 was 3-fold and 12.5-fold attenuated compared to its recombinant and biological parents, respectively (Fig. 3B). At a dose of 5,000 PFU of rPVM-GFP7, the kinetics of weight loss and death were essentially the same as those for the same dose of rPVM (Fig. 3B).

In order to determine the efficiency of replication of the recombinant viruses in vivo, BALB/c mice were inoculated intranasally with 500 PFU of rPVM, rPVM-GFP7, or the biological virus PVM15. Mice were sacrificed on days 3 and 6 after infection, lungs and nasal turbinates were harvested, and virus titers were determined by a plaque assay. For biologically derived PVM15, the mean titer in the lungs was approximately 1 x 105 PFU/g of tissue on day 3 (Fig. 4A) and approximately 4 x 106 PFU/g on day 6 (Fig. 4B). In comparison to PVM15, the mean titer of rPVM in the lungs was reduced threefold (approximately 4 x 104 PFU/g) and twofold (2 x 106 PFU/g) on days 3 and 6, respectively, reflecting the delayed onset of disease and lethality observed in Fig. 3. In the nasal turbinates on day 3, virus was detectable in three out of five mice infected with PVM15 and in just one mouse infected with rPVM (Fig. 4C), likewise indicating a delayed replication of rPVM. In contrast, on day 6, both viruses replicated to approximately the same titer in the nasal turbinates, reaching average titers of approximately 2 x 105 PFU/g of tissue (Fig. 4D).


Figure 4
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  		FIG. 4. Replication of rPVM and rPVM-GFP7 in the upper and lower respiratory tracts of BALB/c mice. Mice were inoculated intranasally with 500 PFU of PVM15, rPVM, or rPVM-GFP7. Mice were sacrificed on day 3 (A and C) or day 6 (B and D) after infection, lungs (A and B) and nasal turbinates (NT) (C and D) were removed and homogenized, and viral titers were determined by a plaque assay. The mean virus titers and the percentages of animals with detectable virus are given below the corresponding graphs. Neighboring groups (indicated with brackets) were compared using the Mann-Whitney test. Asterisks indicate statistically significant differences (P < 0.05). The results of two independent experiments were combined in each graph.

In animals infected with rPVM-GFP7, virus was detected on day 3 in the lungs of only half of the animals and was not detected in the nasal turbinates of any of the animals (Fig. 4A and B). On day 6, the mean titer of rPVM-GFP7 in the lungs was approximately sevenfold reduced compared to that for its parent, rPVM (Fig. 4B), whereas there was no significant difference in the nasal turbinates. In a second experiment, we compared the replication of rPVM and rPVM-GFP7 following intranasal infection with a higher dose, 5,000 PFU (data not shown). At this higher dose, virus was detectable in the nasal turbinates and lungs of all animals on days 3 and 6: the mean viral titer of rPVM-GFP7 was threefold reduced compared to that of rPVM on day 3, whereas the titers were essentially the same on day 6. Thus, these data confirm that rPVM-GFP7 is modestly more attenuated than its parent, rPVM, which in turn is modestly more attenuated than its biological parent, PVM15. These differences were greater on day 3 than on day 6.

Construction and replication of rPVM-GFP-{Delta}G and pPVM-GFP-Gt. We constructed two derivatives of pPVM-GFP7 in which either the complete G gene was deleted ({Delta}G) or the sequence encoding the G cytoplasmic tail was deleted (Gt) (Fig. 5B). The second construct was designed based loosely on the predicted G gene of the nonpathogenic isolate of strain 15 (Fig. 5A). However, instead of introducing a nucleotide insert and frameshift, we deleted 167 nt spanning the beginning of the ORF so as to move the AUG that normally is located at codon 34 (nt 182 to 184) into position as the first AUG in the G ORF. The resulting viruses, rPVM-GFP-{Delta}G and rPVM-GFP-Gt, were readily recovered, and the deletions were confirmed in the viral RNA of the final virus stocks by RT-PCR amplification and sequencing. In addition, an antiserum specific for the PVM G protein confirmed the expression of G by rPVM-GFP-Gt and the lack of G expression in cells infected with rPVM-GFP-{Delta}G (Fig. 5C).


Figure 5
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  		FIG. 5. rPVM-GFP7 viruses with mutations in the G gene. (A) Schematic diagrams (not drawn to scale) of the G genes of the biologically derived virulent and nonpathogenic isolates of PVM strain 15 (23, 34). Open rectangles, ORFs. The amino acid length of the encoded protein is given in each rectangle. Translational start (filled triangles pointing right) or stop (filled vertical bars) codons are identified by their nucleotide positions in the respective G gene. The GS and GE signals are indicated, and nontranslated gene regions are shown as thin horizontal lines. The nucleotide sequence (negative sense) of positions 166 to 190 of the G gene is given, and the inserted U residue that results in a frameshift is shown in parentheses. The complement of the translational stop codon (nucleotides 188 to 190) that is accessed by the frameshift is italicized, and the complement to the alternative start codon at nucleotides 183 to 185 (Met-34) is boldfaced. (B) Schematic representations (not drawn to scale) of rPVM-GFP-{Delta}G and rPVM-GFP-Gt. The diagram in the middle represents the rPVM-GFP7 genome (filled box, GFP gene). The box above the diagram illustrates the deletion of the entire G gene to create rPVM-GFP-{Delta}G. The nucleotide sequence (negative sense) shows the GFP/F gene junction and the AgeI site (italicized) left following the 1,350-nt deletion. The sequence is numbered according to the sequence of rPVM-GFP7, and the GFP GE and F GS signals are underlined. The box below the diagram illustrates the deletion of the cytoplasmic tail of G to create rPVM-GFP-Gt. The unmodified G gene is depicted as in panel A, with the nucleotide positions of its ends numbered according to the complete rPVM-GFP7 sequence. The two potential translational start codons at positions 83 (Met-1) and 182 (Met-34) relative to the G gene sequence are indicated. The nucleotide sequence (negative sense) shows the G GS signal (underlined), the 167-nt deletion that deletes the cytoplasmic tail, and the UAC triplet at positions 182 to 184 that is the complement of the new translational initiation site (Met-34 in the original G ORF). (C) Characterization of rPVM-GFP7, rPVM-GFP-Gt, and rPVM-GFP-{Delta}G with respect to the expression of G protein. Vero cell monolayers in six-well plates were infected with serial dilutions of the indicated viruses and incubated at 32°C under a methylcellulose overlay for 5 days. (Upper panels) GFP-expressing foci were photographed without further treatment by using an inverted fluorescence microscope. (Lower panels) This was followed by fixation and staining using a G-specific antiserum as described for the plaque assay procedure. Each pair of upper and lower panels depicts approximately the same field of view. (D) Multistep growth kinetics of rPVM, rPVM-GFP7, rPVM-GFP-{Delta}G, and rPVM-GFP-Gt in BHK-21 cells. Duplicate monolayers were infected at an input MOI of 0.01 PFU per cell. At the indicated days postinfection, the medium supernatants were harvested and flash frozen, and fresh medium was added. Viral titers were determined by a plaque assay. Means from two independent experiments were used to generate the diagram. Error bars, standard errors of the means.

In a multistep growth study with BHK-21 cells, the kinetics of replication and the final virus yield for the {Delta}G and Gt mutants were essentially indistinguishable from those of their immediate parent, rPVM-GFP7, and also were very similar to those of rPVM (Fig. 5D). Thus, the G protein is completely dispensable for PVM replication in BHK-21 cells. In addition, the N-terminal truncation did not detectably interfere with viral replication, which is consistent with previous findings for similar HRSV mutants in Vero cells (40, 41).

Analysis of G protein expression by rPVM. To investigate whether the G protein of PVM is expressed in a secreted form analogous to that of HRSV, BHK-21 cells were infected with rPVM-GFP7, rPVM-GFP-Gt, or rPVM-GFP-{Delta}G. As a positive control for detecting a soluble G protein, HEp-2 cells were infected with HRSV and treated in parallel. Virus particles and soluble proteins in the medium supernatants were separated and subjected to SDS-PAGE and Western blot analysis as described in Materials and Methods. HRSV proteins were detected using polyclonal antibodies directed against the complete virus (Fig. 6C). In the gel pattern representing purified HRSV, this antiserum reacted with most of the major structural proteins, including the G protein (Fig. 6C, lane V). In the pattern representing the medium supernatant from which the virus had been removed by centrifugation, this antiserum reacted with a single band that migrated slightly more rapidly than virion-associated G protein and thus represented secreted HRSV G protein (Fig. 6C, lane S). The absence of other HRSV structural proteins in this lane confirmed that this band was not virion associated. The PVM G protein was detected using a G-specific rabbit antiserum (Fig. 6A) and an antiserum from a mouse that had recovered from PVM infection (Fig. 6B). In the pattern representing purified virus from rPVM-GFP, the G-specific rabbit serum detected a pair of G protein bands (Fig. 6A) that appear to correspond to the two G-specific bands described by Ling and Pringle (25, 26). The convalescent-phase mouse antiserum detected the same two bands as well as several other structural proteins (Fig. 6B). Similar species were detected for the Gt mutant, for which the pair of G-related species migrated slightly more rapidly than that of rPVM-GFP7. The two species of G protein were absent from purified {Delta}G virus, confirming their identities. However, we were not able to detect the PVM G protein in virus-depleted medium from cells infected with the parental virus or with rPVM-GFP-Gt. Thus, in contrast to HRSV, the PVM G protein does not appear to be expressed significantly in a secreted form by wild-type virus, and deletion of the cytoplasmic tail of G also did not result in the significant production of a secreted form.


Figure 6
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  		FIG. 6. (A and B) Western blot analysis of virus-associated and soluble secreted viral proteins. BHK-21 cells were infected with rPVM-GFP7, rPVM-GFP-Gt, or rPVM-GFP-{Delta}G. Virus particles (V) and soluble proteins in the medium overlying the infected cells (S) were collected separately, subjected to SDS-PAGE under nonreducing conditions, and transferred to nitrocellulose membranes. PVM proteins were detected using a G-specific rabbit antiserum (A) or serum from a convalescent mouse (B). (C) As a positive control, HEp-2 cells were infected with HRSV strain A2. Virus particles and soluble proteins were separated in parallel to those of PVM, and proteins were detected using a commercial polyclonal HRSV-specific antibody. V, virus particles; S, supernatant; M, supernatant from mock-infected cells.

Replication and virulence of rPVM-GFP-{Delta}G and rPVM-GFP-Gt in vivo. In order to evaluate the effect of deletion or truncation of the G protein on PVM replication in vivo, BALB/c mice in groups of five were infected intranasally with rPVM-GFP-{Delta}G or rPVM-GFP-Gt at 500 PFU or 5,000 PFU per mouse. For comparison, one group of three mice received 500 PFU of the parental virus rPVM-GFP7, a dose corresponding to the LD50. The mice were sacrificed on day 6, the lungs and nasal turbinates were removed, and virus titers were determined by a plaque assay (Table 1). In order to determine the virulence of the G mutants, BALB/c mice were infected with doses identical to those described above and were observed closely and weighed daily (Fig. 7).


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  		TABLE 1. Replication of rPVM-GFP-{Delta}G and rPVM-GFP-Gt in the upper and lower respiratory tracts of BALB/c mice


Figure 7
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  		FIG. 7. Virulence of rPVM-GFP-{Delta}G and rPVM-GFP-Gt in mice, measured by changes in body weight. Six- to eight-week-old BALB/c mice were infected intranasally with 500 PFU of rPVM-GFP7 (eight mice), rPVM-GFP-{Delta}G (nine mice), or rPVM-GFP-Gt (nine mice) (A), 5,000 PFU of rPVM-GFP-{Delta}G or rPVM-GFP-Gt (five mice per group) (B), or 5,000 or 50,000 PFU of rPVM-GFP-Gt (five mice per group), as indicated (C). Mice were observed closely and weighed daily. Weights are expressed relative to the weight at day 0, taken as 100%. When necessary, mice that met the end point regulations of the German animal protection board were euthanized by cervical dislocation. The percentage of dead animals per group is given in parentheses in each graph. For panel A, two independent experiments involving four and five animals per group, respectively, were combined.

Although deletion of the complete G gene had no effect on virus replication in vitro, no virus was detected in the nasal turbinates and lungs of mice 6 days after infection with 500 PFU or 5,000 PFU of the {Delta}G virus (Table 1). In contrast, the Gt mutant replicated with an efficiency that was indistinguishable from that of its parent, rPVM-GFP7, resulting in virus titers of 106.1 PFU/g and 106.4 PFU/g in the lungs of mice infected with 500 PFU and 5,000 PFU of rPVM-GFP-Gt, respectively, compared to 106.0 PFU/g after infection with 500 PFU of rPVM-GFP7 (Table 1). Similarly, the Gt virus replicated to the same high titer as its parent, rPVM-GFP7, in the nasal turbinates (Table 1).

Whereas infection with 500 PFU of rPVM-GFP7 resulted in dramatic weight loss and 50% mortality (Fig. 3B and 7A), and infection with 5,000 PFU of rPVM-GFP7 was uniformly lethal (Fig. 3B), the same doses of the {Delta}G or Gt mutant appeared to be benign. Specifically, infection with 500 PFU of the {Delta}G or Gt mutant did not result in any loss of body weight (Fig. 7A) or in the appearance of other disease signs (data not shown). The lack of virulence associated with {Delta}G was not surprising given the lack of detectable virus replication, but the lack of virulence associated with Gt was unexpected given that it appeared to replicate as efficiently as its parent, rPVM-GFP7. After infection with 5,000 PFU of the {Delta}G or Gt mutant, the mice also appeared to be mostly disease free (Fig. 7B). However, in the case of the Gt mutant, there appeared to be a minor weight loss of less than 10% on days 7 and 8 after infection (Fig. 7B). To further investigate possible residual virulence, additional mice in groups of five were infected with 5,000 or 50,000 PFU of the Gt mutant (Fig. 7C). This confirmed that infection with 5,000 PFU resulted in a slight dip in body weight, occurring at day 7 in this case, whereas the 50,000-PFU dose resulted in significant weight loss that started on day 5 after infection and reached a maximum loss in excess of 20% on day 9. The mice showed ruffled fur and reduced activity, although all of the mice recovered.

To estimate the difference in virulence between rPVM-GFP7 and its Gt derivative, we noted that 500 PFU of the former resulted in weight loss approaching 30%, with only 50% of the mice surviving (Fig. 3 and 7A), whereas 50,000 PFU of the latter caused weight loss approaching 30% but did not result in any deaths. This comparison indicates that the Gt mutant was more than 100-fold attenuated compared to its immediate parent, rPVM-GFP7. This was not associated with any difference in replication efficiency or virus load.


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DISCUSSION
 
We recently showed that the isolate of PVM strain 15 that is available from the ATCC is highly virulent in mice and thus resembles the description of the original strain 15 isolate of Horsfall and Hahn (21-23). We determined the complete consensus sequence of the genome of this virus (24) and, in the present study, used it to develop a reverse genetic system. This system was used to investigate the contribution of the viral G protein to replication and virulence in the BALB/c mouse, a convenient experimental animal that is thought to be a natural host and is highly permissive to PVM replication and disease.

Although rPVM retained a high level of virulence in mice, it was slightly attenuated compared to its biological parent with respect to replication and virulence. Specifically, rPVM was reduced twofold in replication in the lower respiratory tract and was fourfold less virulent as measured by the LD50. However, it should be noted that all of the fatalities after infection with 50 PFU of PVM15 were euthanized in response to severe weight loss as required by the German regulations for animal rights, and thus, none of the animals died as a direct consequence of infection. If nature had been allowed to take its course, some of these animals might have survived, and the difference between the biological and recombinant versions might thus have been reduced (although there still would be a difference).

There are several possible explanations for the modest reduction in the virulence of rPVM compared to its biological parent. The recombinant virus differed from its biological parent by the addition of two marker restriction sites, an AgeI site in the SH-G intergenic region and a BstBI site in the M2-L intergenic region, which in each case changed the length of the intergenic region in addition to introducing nucleotide substitutions. While the pneumovirus intergenic regions usually appear to be nonspecific spacers, modest effects on virus gene expression and replication due to manipulation of these sequences cannot be ruled out. A second possibility is that the consensus sequence that we determined might be a composite of more than one virus present in the preparation rather than a single predominant virus, a composite that is slightly suboptimal. It is also possible that this preparation of rPVM acquired one or more adventitious mutations during recovery and passage, although we note that the virus preparation used in this study had been propagated only three times in BHK-21 cells and thus was not a high-passage stock. In any event, the difference in virulence was marginal, and further work will help determine the basis for this modest difference.

The reverse genetics system was used to generate rPVM-GFP7, which expresses GFP. Insertion of this additional gene, which increased the length of the genome by 752 nt (5%), resulted in sevenfold and threefold attenuation of in vivo replication and virulence, respectively, compared to its immediate parent, rPVM. A comparable GFP insert in HRSV was not attenuating in vitro (47), and multiple gene insertions into HMPV that increased its size by 30% only moderately attenuated replication efficiency (5). However, the replication efficiencies of those viruses have not been evaluated in vivo. It was somewhat surprising to find that the addition of another gene to PVM had such a small effect on replication in a highly permissive host. The GFP backbone will represent our "wild-type" virus in future studies, permitting direct fluorescence tracking and histological characterization of pulmonary infection in the natural host.

Subsequently, we used the GFP-bearing backbone to recover rPVM lacking the G protein altogether or encoding a G protein lacking its N-terminal cytoplasmic tail. The latter mutation was based on the G protein of the cell culture-adapted nonpathogenic variant of PVM strain 15 described by Rhandhawa and colleagues (34). The G ORF of this biologically derived variant contained one added nucleotide, causing a frameshift such that the main ORF presumably initiated at the internal AUG at position 183 (Met-34). In that study, expression of a truncated protein of the predicted size was confirmed in a cell-free translation system programmed with synthetic mRNA, and surface expression was confirmed by immunofluorescence of cells expressing a cDNA of the gene (34). In the present study, we simplified the situation by making a deletion that placed Met-34 as the first AUG in the sequence.

Neither mutation—deletion of the entire G gene or deletion of the cytoplasmic tail of G—affected viral replication in cell culture. This is consistent with previous studies in which deletion of the G protein of HMPV had no effect on replication in vitro (6). Somewhat different results were obtained with avian metapneumovirus, in which deletion of the G gene (in combination with the SH gene) attenuated the virus in vitro (30), and HRSV, in which deletion of the G gene was attenuating in human HEp-2 cells but not in African green monkey Vero cells (41).

In contrast to the situation in vitro, rPVM-GFP-{Delta}G was severely attenuated for replication in mice, such that no infectious virus was detected in the lungs or nasal turbinates 6 days after intranasal infection with 500 PFU or 5,000 PFU. However, following infection with the latter (but not the former) dose, mice were protected against challenge 28 days later with a lethal dose of biological wild-type PVM (data not shown). This suggests that a low level of replication occurred, since we have previously observed that inoculation of rodents with 5.7 log10 50% tissue culture infective doses of HMPV was not immunogenic unless the virus was capable of replication (8). The high degree of attenuation due to the deletion of G is comparable to that observed with HRSV {Delta}G in mice (41), whereas a comparable HMPV {Delta}G mutant appeared to be less attenuated in rodent and nonhuman primate models (4). However, the latter models involved infection with significantly (up to 103-fold) higher doses, making a direct comparison difficult.

The Gt virus appeared to replicate as efficiently as its parent, rPVM-GFP7, both in the nasal turbinates and in the lungs, reaching titers in excess of 5.0 log10 and 6.0 log10 PFU/g in the respective tissues. Despite this efficient growth, infection with the Gt virus was asymptomatic at doses that were highly pathogenic and often lethal with its parent, rPVM-GFP7. The Gt virus appeared to be at least 100-fold attenuated compared to its parent, rPVM-GFP7, with respect to weight loss; attenuation based on the LD50 was not available, because none of the animals infected with this virus died under the conditions of these experiments. Thus, the magnitude of virulence was largely dissociated from the magnitude of replication. This dissociation was not complete, since increasing the dose of the Gt virus to 50,000 PFU resulted in weight loss, and it seems likely that a higher dose would result in deaths. Nonetheless, whereas the parent, rPVM-GFP7, induced weight loss at doses of 500 and 5,000 PFU and was 50% and 100% lethal, respectively, at these doses, its Gt derivative was essentially nonpathogenic at the same doses. To our knowledge, this is the first example of an attenuating mutation in a pneumovirus whose effect is not mediated primarily by reduced viral replication. This is offered with the caveat that we did not determine virus titers on every day between inoculation and day 10, when deaths began to occur with the rPVM-GFP7 virus. Nonetheless, the equivalency between rPVM-GFP-Gt and pPVM-GFP7 at the peak of replication on day 6 suggests that the former virus replicated with an efficiency similar to that of its parent.

In the present study, we were not able to detect a secreted form of the PVM G protein in the supernatants of cells infected with PVM expressing the wild-type or truncated G protein. This is consistent with the surface expression of truncated G observed by Rhandhwa and colleagues, as mentioned above (34), although that study did not address whether a proportion of wild-type or truncated G protein might also be secreted. This is in contrast with HRSV, for which a substantial proportion of G protein expressed from the wild-type gene is secreted. In the case of HRSV, secretion is dependent on translational initiation at the second AUG in the ORF (Met-48), which is located within the transmembrane anchor, followed by proteolytic trimming that completely removes the remaining transmembrane domain and creates a new N terminus at position 66 (19). In the case of PVM, the G ORF contains a second AUG at codon 34, but it is in a sequence context that is not consistent with efficient translational initiation due to the presence of a C in the –3 position, and it is not known whether significant translation occurs. In addition, as already noted, the second AUG in the PVM G ORF is located at the inner face of the transmembrane domain rather than in the middle of this domain. In any event, no secreted form of PVM G was observed. Thus, while the immunomodulatory function of the HRSV G protein appears to depend in large part on its secretion (32), the effects of PVM G presumably are not mediated by a comparable species.

These findings suggest that the cytoplasmic tail of G has characteristics of a true virulence factor, that is, a factor whose pathogenic effect can be dissociated from pathogen load. The mechanism of attenuation for the Gt virus remains unknown. Comparisons between the biologically derived nonpathogenic version of strain 15 and the virulent strain J3666 indicated that the pathogenesis observed with the latter was associated with enhanced pulmonary inflammation that was paralleled by increased induction of proinflammatory cytokines, including macrophage inflammatory protein 1{alpha}, macrophage chemoattractant peptide-1, eotaxin, and others (13). Thus, the virulence of strain J3666 appears to be mediated, at least in part, by an overly robust inflammatory response. As already noted, strains 15 and J3666 contain numerous differences involving a number of proteins and potential cis-acting elements, including residues that are conserved within the Pneumovirus genus (24, 42). Therefore, while it seems likely that the truncation of the G protein will prove to be an important factor in the differences between the pathogenic and nonpathogenic phenotypes characterized in the published comparisons between strains 15 and J3666 (42), this will need to be directly evaluated. If the cytoplasmic tail of G indeed is a major contributor to the difference between an "inflammatory" and a "noninflammatory" phenotype, it will be interesting to determine the mechanism for this effect. One possibility is that the cytoplasmic tail directly affects intracellular signaling. As an inexact precedent, the cytoplasmic tail of pathogenic Hantaan virus recently was found to inhibit the induction of type I interferon involving the RIG-I helicase, whereas the cytoplasmic tail of a nonpathogenic strain lacked this ability (1). In the case of PVM, one possibility is that the cytoplasmic tail somehow stimulates activation of NF-{kappa}B, leading to the expression of proinflammatory cytokines, and that its deletion ablates this ability. However, further studies are necessary to investigate the mechanistic basis for these effects.

Deletion of the cytoplasmic tail provides a novel attenuating mutation for the pneumoviruses. If the attenuation phenotype of the Gt virus can be reproduced in those pneumoviruses for which a vaccine is needed (HRSV, HMPV, avian metapneumovirus), it would be ideal for developing a live vaccine. A particular problem with live vaccines is that attenuation usually is based on decreased virus replication, which in turn reduces immunogenicity. A viral mutant that replicates efficiently without causing disease would be an ideal vaccine candidate, one that combines safety with a high level of immunogenicity.

In conclusion, we have developed a reverse genetic system for PVM that largely reproduces the virulent phenotype of the biological wild-type virus. The utility of the system was shown by construction of recombinant viruses expressing GFP as marker protein, as well as by construction of gene deletion/truncation mutants that investigated the contribution of the G protein to virulence. The capability of engineering recombinant PVM provides a convenient model for characterizing factors involved in pneumovirus virulence in a natural host.


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ACKNOWLEDGMENTS
 
We thank Jürgen Brandel for his expertise in photography and Renate Mielke for technical assistance.

This work was supported in part by the NIAID Intramural Research Program and by a grant from the Ministry of Science, Research and the Arts of Baden-Württemberg, Germany (Az: 21-655.042-1-1/1), to C.D.K.


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FOOTNOTES
 
* Corresponding author. Present address: Institute of Virology and Immunobiology, University of Würzburg, Versbacher Strasse 7, D-97078 Würzburg, Germany. Phone: (49) 931-201-49586. Fax: (49) 931-201-49553. E-mail: ckrempl{at}vim.uni-wuerzburg.de Back

{triangledown} Published ahead of print on 13 June 2007. Back


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Journal of Virology, September 2007, p. 9490-9501, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00364-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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