JoséA. Melero

Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus.

The genetic characterization of four previously reported mutants of human respiratory syncytial (RS) virus resistant to monoclonal antibody 63G is described. Sequences of the G protein genes were obtained from: (i) mRNA derived cDNA recombinants, (ii) direct mRNA sequencing and (iii) amplified vRNA derived cDNAs. The results obtained indicate that the original escape mutants, recovered from individual plaques, contained heterogeneous viral populations. This heterogeneity affected the number of adenosine residues present after nucleotides 588 or 623 of the G protein gene. Mutant viruses recovered after a second plaque purification step generated homogeneous sequences but contained single adenosine insertions or deletions at those two sites compared with the Long sequence. These genetic alterations introduced frameshift changes which are reflected in both the antigenic and structural properties of the mutant G proteins. The origin and importance of frameshift mutations in the RS virus G protein gene are discussed.

Analysis of genetic variability in human respiratory syncytial virus by the RNase a mismatch cleavage method: Subtype divergence and heterogeneity

We have applied the RNase A mismatch cleavage method to the analysis of genetic variability among human Respiratory Syncytial (RS) viruses. Antisense RNA probes of the Long strain were hybridized to total RNA extracted from cells infected with other strains. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products analyzed by gel electrophoresis. Each virus generated characteristic band patterns with the different probes. Comparative analyses of the cleavage patterns indicate that antigenic subtypes correlate with genetically distinct viral groups. Viruses within each subtype, however, show substantial genetic heterogeneity and progressive accumulation of genetic changes with time. This heterogeneity is also observed among viruses of the same epidemic outbreak which cannot be distinguished with a panel of monoclonal antibodies. Different genes and gene regions also differ in their rates of change. These results are discussed in terms of RS virus evolution.

Participation of cytoskeletal intermediate filaments in the infectious cycle of human respiratory syncytial virus (RSV)

RSV infection of Hep-2 or HeLa cells leads to biochemical and morphological changes of cytoskeletal intermediate filaments (IF). Thus, human cytokeratin 18 is modified to generate a more acidic polypeptide of slightly larger apparent molecular weight. In addition, the amounts of vimentin and other cytokeratins are reduced, probably as a consequence of proteolytic degradation. These changes are reflected in a decrease of immunofluorescence with specific antibodies in RSV-induced syncytia and a more disorganized arrangement of IF arrays. About 50% of virus nucleoprotein (NP) is extracted with the high salt and detergent-insoluble intermediate filament fraction. Pulse-chase experiments indicate that NP needs a maturation period after synthesis to associate with IF. It is suggested that RSV needs to interact with IF during its life cycle and that association of NP, and/or other viral components, with IF might then lead to cytoskeletal structures becoming unstable in RSV-infected cells.

Nuclear transport of influenza virus polymerase PA protein

The subcellular distribution of influenza polymerase PA subunit has been studied using a SV40-recombinant virus (SVPA76), which allows the expression and accumulation of this protein in COS-1 cells. In contrast to the complete nuclear localization observed for the PA subunit several hours after influenza virus infection, when COS-1 cells were infected with the SVPA76 recombinant, the PA protein accumulated either in the nucleus, in the cytoplasm or was distributed throughout the cell. When cells were infected with the SVPA76 recombinant and superinfected with influenza virus, a clear increase in the proportion of cells showing nuclear localization of the PA protein was observed, suggesting that some trans-factor may be required to allow complete nuclear accumulation of the protein. Double infections using SVPA76 recombinant and either SVPB1 or SVNS recombinant viruses showed a complete correlation between expression of polymerase PB1 subunit or NS1 protein and nuclear localization of polymerase PA subunit. However, no such correlation was observed in the double infections of SVPA76 and SVNP recombinants. These results suggest that polymerase PB1 subunit and the non-structural proteins could be involved in the nuclear targeting or nuclear retention of influenza polymerase PA protein.

Isolation of cross-reactive, subtype-specific monoclonal antibodies against influenza virus HA1 and HA2 hemagglutinin subunits

SummaryThe large (HA1) and small (HA2) subunits of influenza virus A/Vict/3/75 hemagglutinin were purified in denatured form by preparative electrophoresis. Both polypeptides were used to immunize mice from which monoclonal antibodies were obtained. These antibodies reacted not only with the corresponding hemagglutinin subunit but also with purified virions. When tested by radioimmunoassay against a panel of human viruses, most anti-HA1 and -HA2 antibodies behaved as subtype-specific, whereas anti-HA antibodies, raised against purified virus, were more restricted. The anti-subunit antibodies were negative in hemagglutination-inhibition and neutralization tests. The interest of these antibodies as reagents for research and diagnosis is discussed.

Epitope mapping of cross-reactive monoclonal antibodies specific for the influenza A virus PA and PB2 polypeptides

Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti-PB2 MAbs, respectively. Five of the anti-PA MAbs recognized antigenic determinants located within the amino-terminal 157 amino acids of the PA protein, and 6 others reacted strongly with a PA fragment comprising the first 236 amino acids. All 8 anti-PB2 antibodies reacted strongly with a polypeptide fragment containing amino acids 1-113 of the PB2 protein. Analyses of the reactivities of 4 anti-P antibodies with 23 influenza A virus reference strains isolated over a period of 61 years and recovered from humans, pigs, birds and horses, showed that the epitopes were conserved among all viral isolates. The application of these antibodies as research and diagnostic tools is discussed.

Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes.

Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non‐structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T‐antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift‐down, defined circular DNA molecules were generated and amplified up to 2000‐5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20‐65‐fold in the expression of NS1 and NS2 proteins was observed after temperature shift‐down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes.

Conservation of epitopes recognized by monoclonal antibodies against the separated subunits of influenza hemagglutinin among type A viruses of the same and different subtypes

SummaryMonoclonal antibodies raised against the separated hemagglutinin subunits (HA1 and HA2) of influenza A/Vic/3/75 (H3N2) virus were tested against a large panel of human and avian strains. The epitopes recognized by most antibodies were conserved among subtype H3 viruses, but reactivity of some antibodies with members of other subtypes was also observed. Particularly, the H4 virus reacted with most antibodies directed against the HA2 subunit. These results are discussed in terms of sequence similarities between subtypes and application of these antibodies as subtyping reagents.

Reduced expression of surface glycoproteins in mouse fibroblasts persistently infected with human respiratory syncytial virus (HRSV)

Summary. BCH4 cells, persistently infected with Human Respiratory Syncytial Virus (HRSV), were obtained by Fernie et al. [12] after infection of a BALB/c mouse embryo cell line with the Long strain of HRSV. To understand the basis of HRSV persistence, the expression of HRSV RNAs and proteins was evaluated in BCH4 cells and infected parental BALB/c and fully permissive HEp-2 cells. Production of viral mRNAs was severely impaired in BCH4 cells. In addition, the expression level of the surface glycoproteins F and G was markedly reduced relative to internal viral proteins. However, virus recovered from BCH4 cells could lytically infect HEp-2 cells and expressed normal levels of surface glycoproteins. No evidence of defective genomes or interfering particles was found in BCH4 cells. Taken together, these data indicate that reduction of both viral mRNA accumulation and surface glycoprotein biosynthesis are at the basis of HRSV persistence in BCH4 cells.