Human metapneumovirus (HMPV) has recently been identified as a significant cause of serious respiratory tract disease in humans. In particular, the emerging information on the contribution of HMPV to pediatric respiratory tract disease suggests that it will be important to develop a vaccine against this virus for use in conjunction with those being developed for human respiratory syncytial virus and the human parainfluenza viruses. A recently described reverse genetic system (S. Biacchesi, M. H. Skiadopoulos, K. C. Tran, B. R. Murphy, P. L. Collins, and U. J. Buchholz, Virology 321:247-259, 2004) was used to generate recombinant HMPVs (rHMPVs) that lack the G gene, the SH gene, or both. The DeltaSH, DeltaG, and DeltaSH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Thus, the SH and G proteins are not essential for growth in cell culture. Apart from the absence of the deleted protein(s), the virions produced by the gene deletion mutants were similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. When administered intranasally to hamsters, the DeltaG and DeltaSH/G mutants replicated in both the upper and lower respiratory tracts, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were at least 40-fold and 600-fold restricted in replication in the lower and upper respiratory tract, respectively, compared to wild-type rHMPV. They also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Surprisingly, G is dispensable for protection, and the DeltaG and DeltaSH/G viruses represent promising vaccine candidates. In contrast, DeltaSH replicated somewhat more efficiently in hamster lungs compared to wild-type rHMPV (20-fold increase on day 5 postinfection). This indicates that SH is completely dispensable in vivo and that its deletion does not confer an attenuating effect, at least in this rodent model.

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A wide variety of RNA viruses have been shown to produce proteins that inhibit interferon (IFN) production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to block IFN signaling by causing the proteasomal degradation of STAT2. In addition, recombinant RSVs lacking either NS1 or NS2 induce more IFN production than wild-type (wt) RSV in infected cells. However, the mechanisms by which the NS proteins perform this function are unknown. In this study, we focused on defining the mechanism by which NS2 inhibits the induction of IFN transcription. We find that NS2 is required for the early inhibition of IFN transcription since the infection of cells with NS2-deletion RSV resulted in a higher level of IRF3 activation at early time points postinfection compared with that of wt or NS1-deletion RSV infection. In addition, NS2 expression inhibits IFN transcription induced by both the RIG-I and TLR3 pathways. Furthermore, we show that NS2 inhibits RIG-I-mediated IFN promoter activation by binding to the N-terminal CARD of RIG-I and inhibiting its interaction with the downstream component MAVS (IPS-1, VISA, Cardif). Thus, the RSV NS2 protein is a multifunctional IFN antagonist that targets specific components of both the IFN induction and IFN signaling pathways.Respiratory syncytial virus (RSV) is the most important etiologic agent of pediatric viral respiratory infection and remains a major cause of morbidity and mortality among infants as well as immunocompromised subjects and the elderly (9). RSV is the prototype member of the Pneumovirus genus in the family Paramyxoviridae. The nonsegmented, negative-sense RNA genome of RSV encodes 10 transcription units from which 11 proteins are translated. From this linear array, the two promoter-proximal genes encode the nonstructural NS1 and NS2 proteins which are dispensable for viral replication in vitro, although the deletion of either NS gene attenuates the replication of recombinant RSV (rRSV) significantly in vitro and markedly in vivo (7,16,32,39,42). Since the NS genes are the first two transcription units in the gene order, the transcription of NS1 and NS2 is thought to be abundant and to occur early in infection (9).The RSV NS proteins are small (NS1, 139 amino acids [aa]; NS2, 124 aa) and have no significant sequence homology with each other or with any cellular protein in the database. NS1 and NS2 have been shown to encode multiple functions associated with viral pathogenesis. NS1 and NS2 appear to antagonize both the cellular antiviral response as well as the induction of interferon (IFN) transcription (5,6,32,36). rRSVs lacking NS1 (⌬NS1) or NS2 (⌬NS2) induce significantly more IFN-␤ transcription in infected cells than wild-type (wt) rRSV; the deletion of both NS genes from rRSV results in a virusinduced IFN-␤ transcription to a greater extent than either single NS deletion rRSV (36). In addition, RSV infection results in the proteasome-mediated degradation of STAT2 by the formation of a ubiquitin ligas...

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, encodes a small hydrophobic (SH) protein of unknown function. Parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, also encodes an SH protein, which inhibits tumor necrosis factor alpha (TNF-␣) signaling. In this study, recombinant PIV5 viruses without their own SH but containing RSV SH (from RSV strain A2 or B1) in its place (PIV5⌬SH-RSV SH) and RSV lacking its own SH (RSV⌬SH) were generated and analyzed. The results indicate that the SH protein of RSV has a function similar to that of PIV5 SH and that it can inhibit TNF-␣ signaling.Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children (17). RSV, along with the prototype paramyxovirus parainfluenza virus 5 (PIV5; formerly known as simian virus 5), is a member of the Paramyxoviridae family, which includes important human and animal pathogens. Both RSV and PIV5 encode small hydrophobic (SH) proteins, which are type II transmembrane proteins. The SH protein of RSV contains 64 (RSV subgroup A) or 65 (RSV subgroup B) amino acid residues (Fig. 1A) (3)(4)(5)14). Some studies have suggested that the RSV SH protein may have a role in viral fusion (9, 19) or in changing membrane permeability (15). However, RSV lacking the SH gene (RSV⌬SH) is viable, causes syncytium formation, and grows as well as the wild-type virus (1, 10, 11), indicating that the SH protein is not necessary for virus entry into host cells or syncytium formation (19). RSV⌬SH is attenuated in animals, indicating that RSV plays an important role in viral pathogenesis (1). Interestingly, recombinant PIV5 lacking the SH gene (rPIV5⌬SH) has a similar phenotype: it has normal growth in vitro, but it is attenuated in vivo (7). Studies of rPIV5⌬SH have shown that the SH protein is necessary for the inhibition of tumor necrosis factor alpha (TNF-␣)-induced apoptosis in L929 cells (12). Recent work suggests that the SH protein of mumps virus is a functional counterpart of the PIV5 SH protein (22), even though the PIV5 and mumps SH proteins have no sequence homology. We hypothesized that the SH protein of RSV may be functionally similar to other SH proteins from members of the Paramyxoviridae family. To test this hypothesis, recombinant viruses that contained the RSV SH gene of strain A2 or B1 in place of the PIV5 SH gene were produced and confirmed by reverse transcription (RT)-PCR (Fig. 1B). The rPIV5 and rPIV5⌬SH viruses grow to similar titers, although rPIV5⌬SH virus grows slightly faster in the first stages of infection (Fig. 1C) (6, 22). Growth of the rPIV5⌬SH-RSV SH recombinant viruses was comparable to that of rPIV5 and rPIV5⌬SH up to 2 days postinfection (dpi). Occasionally, a delay in the growth of one or both of the recombinant viruses was observed, but by 24 or 36 h the viruses had always reached titers comparable to that of the wild-type virus (Fig. 1C). The plaques formed by the rPIV5, rPIV5⌬SH, and rPIV5⌬SH-RSV SH viruses in BHK cells were of a...

Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.