Human respiratory syncytial virus (HRSV), a member of the pneumovirus subfamily of the paramyxovirus family, is an enveloped RNA virus that is the most important viral agent of pediatric respiratory tract disease worldwide. Human metapneumovirus (HMPV), first identified in 2001, is a second human pneumovirus that also is a significant pediatric pathogen. For HRSV, we previously determined a complete, functional sequence of genomic RNA, mapped its genes, identified its encoded proteins, determined the extent of naturally-occurring sequence diversity, and developed a reverse genetic system for producing infectious HRSV entirely from cloned cDNAs. This analysis has now been extended to HMPV. Complete consensus nucleotide sequences were determined for human metapneumovirus (HMPV) isolates CAN97-83 and CAN98-75, representing the two proposed genotypes or genetic groups of HMPV. The overall level of genome nucleotide sequence identity and aggregate proteome amino acid sequence identity between the two HMPV groups were 80% and 90%, respectively, similar to the respective values of 81% and 88% between the two antigenic subgroups of human respiratory syncytial virus (HRSV). The relatedness between HMPV groups was least for the SH and G proteins (59% and 37% identity, respectively), which were even more divergent than their HRSV counterparts (72% and 55% cross-subgroup identity, respectively). Thus, the G protein, a putative protective antigen, is highly divergent between the two HMPV genetic groups. The F protein, a second putative protective antigen, was highly conserved between the HMPV groups and likely would induce cross-protective immunity. We conclude that the two genetic groups of HMPV are approximately comparable in genetic diversity to the two antigenic subgroups of HRSV. We developed a reverse genetic system for the CAN97-83 isolate, by which complete infectious HMPV can be recovered by coexpression of a set of cloned cDNAs separately encoding a positive-sense copy of the viral genome and the following HMPV proteins: the nucleocapsid N, phosphoprotein P, polymerase protein L, and putative nucleocapsid-associated protein M2-1. We designed a version of HMPV, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette placed 58 nt from the 3' end of the genome. 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 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. We also recovered a version of HMPV without the added GFP gene. This virus replicated in vitro as efficiently as biologically-derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in the promoter proximal position in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb in length, 30% longer than that of the natural virus. This 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. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes in order to increase gene dose or to provide representation of additional antigenic lineages or subgroups of HMPV. We used a reverse genetic system for HRSV to study the role of the transcriptional termination or gene-end (GE) signals in mononegavirus transcription. For this type of virus, gene expression appears to be regulated primarily by a polar transcriptional gradient governed by polymerase fall-off at the intergenic junctions. In addition, naturally occurring differences in the transcription termination efficiency (TE) by the various GE signals due to variable nucleotides provide the possibility of an additional level of regulation. We made a series of mutations in the GE signals of the NS1 and/or NS2 genes of HRSV that increased or decreased their TE. These genes were chosen because their naturally occurring GE signals are very inefficient at transcriptional termination, suggesting that this feature might have biological significance. Also, they are the first two genes in the transcriptional map, and hence changes in their TE should have the greatest impact on overall gene transcription. We incorporated these changes into recombinant virus and monitored the effect of these changes on expression of each gene, the expression of their immediate downstream neighbor(s), expression of genes that were further downstream in the transcriptional map, and the efficiency of viral growth in vitro and in mice. Decreases in TE resulted in reduced synthesis of monocistronic mRNA from the gene in question and its immediate downstream neighbor, and a concomitant increase in readthrough transcription. This is indicative of an intercovertibity between monocistronic and readthrough mRNAs mediated by the GE signals. Increases in TE had the converse effect. These changes were found to have essentially no effect on the transcription of genes further downstream, indicating that TE does not make a significant contribution to controlling the amount of polymerase that moves down the genome. Each of the mutant viruses displayed growth kinetics and virus yields similar to wild-type recombinant RSV (rA2) in vitro, and each grew similarly to rA2 in the upper and lower respiratory tract of BALB/c mice, though some of the mutants displayed slightly decreased replication. These data suggest that the natural inefficiencies of transcription termination by the NS1 and NS2 GE signals do not play important roles in controlling the magnitude of RSV gene expression or the efficiency of virus replication. We investigated the possibility of intermolecular RNA recombination for HRSV, which had not been previously documented for any mononegavirus. Cells were coinfected with two rRSV mutants, one lacking the G gene (deltaG/HEK) and the other the NS1 and NS2 genes (deltaNS1/2). These viruses replicate inefficiently and form pinpoint plaques in HEp-2 cells. Therefore, potential recombined viruses with a growth and/or plaque formation advantage should easily be identified and differentiated from the two parental viruses. In one of six co-infections, an HRSV variant was detected and identified as a recombined HRSV (rec-HRSV). The rec-HRSV appeared to have been generated by a polymerase jump from the deltaG/HEK genome to that of deltaNS1/2 and back again in the vicinity of the SH-G-F genes. This apparently involved nonhomologous and homologous recombination events, respectively, The recombined genome was identical to that of the deltaG/HEK mutant except that all but the first 12 nucleotides of the SH gene were deleted and replaced by an insert consisting of the last 91 nucleotides of the G gene and its downstream intergenic region. This resulted in the formation of a short chimeric SH:G gene. Northern and Western blot analysis confirmed that the rec-HRSV did not express the normal SH and G mRNAs and proteins but did express the aberrant SH:G mRNA. This provides an experimental demonstration of intermolecular recombination yielding a viable, helper-independent mononegavirus. However, the isolation of only a single rec-HRSV under these optimized conditions supports the idea that RNA recombination is rare indeed and not problematic for vaccine development.
Showing the most recent 10 out of 15 publications
