Frequent Frameshift and Point Mutations in the SH Gene of Human Metapneumovirus Passaged In Vitro

Frequent Frameshift and Point Mutations in the SH Gene of Human Metapneumovirus Passaged In Vitro,10.1128/JVI.00128-07,Journal of Virology,Stephane Bi

Frequent Frameshift and Point Mutations in the SH Gene of Human Metapneumovirus Passaged In Vitro   (Citations: 5)
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During the preparation of recombinant derivatives of the CAN97-83 clinical isolate of human metapneu- movirus (HMPV), consensus nucleotide sequencing of the recovered RNA genomes provided evidence of frequent sequence heterogeneity at a number of genome positions. This heterogeneity was suggestive of sizable subpopulations containing mutations. An analysis of molecularly cloned cDNAs confirmed the presence of mixed populations. The biologically derived virus on which the recombinant system is based also contained sizeable mutant subpopulations, whose presence was confirmed by biological cloning and nucleotide sequenc- ing. Most of the mutations occurred in the SH gene. For example, partial consensus sequencing of 40 independent preparations of recombinant HMPV (wild-type and various derivatives) showed that 31 of these preparations contained a total of 41 instances of small insertions in the SH gene and a total of five small insertions elsewhere. In each of these 31 preparations, there was at least one insert in SH that changed the reading frame and would yield a truncated protein. Nearly all of these insertions involved adding one or more A residues to various tracks of four or more A residues, with the most frequent site being a tract of seven A residues. There were also two instances of nucleotide deletions and numerous instances of nucleotide substi- tution point mutations, mostly in the SH gene. The occurrence of mutant subpopulations was greatly reduced by the replacement of the SH gene with a synthetic version in which these oligonucleotide tracts were eliminated by silent nucleotide changes. We suggest that we frequently detected subpopulations in which the expression of full-length SH protein was ablated because it provided a modest selective advantage to this clinical isolate in vitro. Adaptation involving the functional loss of a gene is unusual for an RNA virus. Human metapneumovirus (HMPV), which was first de- scribed in 2001 (26), is a member of the Paramyxoviridae family and has been assigned to the Metapneumovirus genus of the Pneumovirinae subfamily, together with avian metapneumovi- rus. Human respiratory syncytial virus (HRSV), the member of the Pneumovirinae that has been studied in the greatest detail, belongs to the second genus of Pneumovirinae, Pneumovirus (10). HMPV is an important cause of respiratory tract infec- tion worldwide, resembling HRSV, and is estimated to be associated with 5 to 15% of pediatric hospitalizations due to respiratory tract infections (13, 18, 27, 28). There presently are no licensed vaccines or specific therapies for HMPV (9). Like all members of the Paramyxoviridae family, HMPV is an enveloped, single-stranded, negative-sense RNA virus. The genome is approximately 13 kb in length, contains eight genes in the order 3-N-P-M-F-M2-SH-G-L-5, and encodes nine proteins, with the M2 mRNA containing two overlapping open reading frames (ORFs) that are expressed as two separate proteins, M2-1 and M2-2 (4, 7, 25). The HMPV proteins are the following: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion glycoprotein; M2-1, putative transcription factor; M2-2, RNA synthesis regulatory factor; SH, small hy- drophobic glycoprotein; G, attachment glycoprotein; and L, viral polymerase. F, G, and SH are transmembrane surface glycoproteins that are packaged in the virus particle. F has been shown to be a major neutralization and protective anti- gen, whereas G and SH appear to have minor and insignificant roles, respectively, in inducing neutralizing antibodies and pro- tection (23). We and others previously developed reverse-genetics sys- tems for HMPV (5, 15), whereby complete infectious virus can be recovered from cultured cells that have been transfected with plasmids encoding a positive-sense copy of the viral ge- nome and the N, P, M2-1, and L proteins. This recombinant system provides the basis for introducing defined mutations into infectious HMPV. In particular, this approach is currently being used to develop attenuated mutants of HMPV for use in a live intranasal vaccine (2, 9, 21). Pertinent to the present report, studies with recombinant viruses showed that the de- letion of SH and G, individually or in combination, had little or no apparent effect on replication in cell culture (6). Virus in which the M2-1 and M2-2 ORFs were silenced separately or in which the entire gene was deleted also replicated in cell culture with an efficiency similar to that of wild-type HMPV, although the deletion of M2-1 appeared to be slightly attenuating (7). The present report describes how, in the course of preparing a large number of independent preparations of various recom- binant viruses, we found evidence of frequent sequence hetero- geneity. Most of these mutations occurred in the SH gene and created frameshifts that would drastically affect protein coding; numerous point mutations also were observed. Comparable frameshift mutations in the also-dispensable G and M2 ORFs were not observed. The frequent expansion of subpopulations
Journal: Journal of Virology - J VIROL , vol. 81, no. 11, pp. 6057-6067, 2007
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