Sequence and structure relatedness of matrix protein of human respiratory syncytial virus with matrix proteins of other negative-sense RNA viruses.

Matrix proteins of viruses within the order Mononegavirales have similar functions and play important roles in virus assembly. Protein sequence alignment, phylogenetic tree derivation, hydropathy profiles and secondary structure prediction were performed on selected matrix protein sequences, using human respiratory syncytial virus matrix protein as the reference. No general conservation of primary, secondary or tertiary structure was found, except for a broad similarity in the hydropathy pattern correlating with the fact that all the proteins studied are membrane-associated. Interestingly, the matrix proteins of Ebola virus and human respiratory syncytial virus shared secondary structure homology.

Association of matrix protein of respiratory syncytial virus with the host cell membrane of infected cells.

The matrix protein of paramyxoviruses plays an important role in virus assembly through its interactions with cell membrane, virus envelope and virus nucleocapsid. In the present study, we investigated the possible association of respiratory syncytial virus (RSV) matrix (M) protein with the plasma membrane of infected cells. Using confocal microscopy we found that M was present at the cytoplasmic side of the plasma membrane. We used flotation gradients to purify membranes from RSV infected cells and treated them with cold Triton X-100 to obtain lipid rafts in the insoluble fraction. Western blot of the lipid raft fraction with specific antibodies showed that it contained M, as well as G (attachment) and N (nucleocapsid) proteins. We also found that RSV purified on sucrose gradients contained lipid raft markers. Together, our data suggest that RSV uses lipid rafts for assembly and budding.

Multiple heparin binding domains of respiratory syncytial virus G mediate binding to mammalian cells.

Respiratory syncytial virus (RSV) G glycoprotein mediates cell attachment through surface glycosaminoglycans (GAGs). Feldman et al. [10] suggested that specific basic amino acids in residues 184-198 of G defined a critical heparin binding domain (HBD). To further define the G HBD we made a series of truncated G proteins expressed in Escherichia coli. G88 (G residues 143-231), bound to HEp-2 cells in a dose dependent manner and binding was inhibited >99% with heparin. Cell binding of G88 was unaltered by alanine substitution mutagenesis of all basic amino acids in Feldman's region 184-198. A G88 variant truncated beyond residue 198, G58, and G58 fully alanine substituted in the region 184-198, G58A6, bound to HEp-2 cells about half as well and 100-fold less well than G88, respectively. G88 and all alanine substitution mutants of G88 inhibited RSV plaque formation by 50% (ID(50)) at concentrations of approximately 50 nM; the ID(50) of G58 was approximately 425 nM while G58A6 had an ID(50) >1600 nM. These data show that the G HBD includes as much as residues 187-231, that there is redundancy beyond the previously described HBD, and that the cell-binding and virus infectivity-blocking functions of these recombinant G proteins were closely linked and required at least one HBD.

The matrix protein of Human respiratory syncytial virus localises to the nucleus of infected cells and inhibits transcription.

We studied the kinetics of localisation of matrix (M) protein of Human respiratory syncytial virus (RSV) in infected cells. M protein was detected in the nucleus early in infection, by confocal microscopy and by immunoblotting of nuclear fractions. We next tested the possibility that M protein may be involved in inhibition of host cell transcription. Nuclear extracts from RSV infected cells had less transcriptional activity in vitro when compared to nuclear extracts from mock infected cells. In addition, nuclear extracts from RSV infected cells inhibited the transcriptional activity of nuclear extracts from mock infected cells, suggesting that an inhibitory activity was transferred with nuclear extracts from RSV infected cells. Our data suggest that M protein may play a role early in the infection by inhibiting host cell transcription.

Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells.

Little is known about the functions of the matrix (M) protein of respiratory syncytial virus (RSV). By analogy with other negative-strand RNA viruses, the M protein should inhibit the viral polymerase prior to packaging and facilitate virion assembly. In this study, localization of the RSV M protein in infected cells and its association with the RSV nucleocapsid complex was investigated. RSV-infected cells were shown to contain characteristic cytoplasmic inclusions. Further analysis showed that these inclusions were localization sites of the M protein as well as the N, P, L and M2-1 proteins described previously. The M protein co-purified with viral ribonucleoproteins (RNPs) from RSV-infected cells. The transcriptase activity of purified RNPs was enhanced by treatment with antibodies to the M protein in a dose-dependent manner. These data suggest that the M protein is associated with RSV nucleocapsids and, like the matrix proteins of other negative-strand RNA viruses, can inhibit virus transcription.

Surfactant protein A binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity.

Collectins are a family of calcium-dependent collagenous lectins that appear to be important in innate host defense. We investigated the ability of three human collectins, namely, lung surfactant proteins A (SP-A) and D (SP-D) and the serum mannose-binding protein (MBP), to bind to the surface glycoproteins of respiratory syncytial virus (RSV). SP-A was shown to bind to the F (fusion) glycoprotein but not to the viral G (attachment) glycoprotein, and binding was completely abrogated in the presence of EDTA. Neither SP-D nor MBP bound to either glycoprotein. SP-A also neutralized RSV in a calcium dependent fashion. These results support a role for SP-A in the defense of infants against infection with RSV and indicate a possible mechanism for its protective activity.

Expression and characterisation of the ovine respiratory syncytial virus (ORSV) G protein for use as a diagnostic reagent.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children and calves. Antibodies to ovine RSV (ORSV) are common in sheep, but the clinical disease is not well defined. There is no report of ORSV infection in Australian sheep although respiratory distress syndrome has been described. This discrepancy may be due to the lack of a suitable diagnostic test. In this report, we have characterised the ORSV G protein in an attempt to study its relatedness to human and bovine RSV (HRSV, BRSV) and for use in the development of a suitable diagnostic assay. Full length and a truncated variant of ORSV G protein were expressed in mammalian cells and the expressed proteins characterised by indirect immunofluorescence and radioimmunoprecipitation assays. Our results indicate that like HRSV, the ORSV G protein is heavily glycosylated. The expressed protein was membrane bound as well as secreted and could be purified from culture supernatants and may be suitable for use in development of a diagnostic assay.

Tissue tropism of avian reoviruses is genetically determined.

Two genome segments, M2 and S1, were preferentially selected in reassortants isolated in Vero cells. Analysis with monoclonal antibodies (MAbs) against RAM-1 strain showed that the 39-kDa protein encoded by the genome segment S1 contained epitopes involved in neutralisation of virus infectivity for both Vero and chicken kidney (CK) cells. The 39-kDa protein appeared to have two major epitopes that are attachment sites for cell receptors, one interacting only with CK cell receptors and the other with both CK and Vero cell receptors but principally Vero cell receptors. These results suggest that the strain RAM-1 may have developed an epitope for Vero cell receptors owing to mutation in the S1 genome segment, but still retained the epitope responsible for infection of CK cells.

Sequence variation within the capsid protein of Australian isolates of feline calicivirus.

The capsid protein of Australian feline calicivirus (FCV) isolates is demonstrably different from the prototype strain F9. Five Australian isolates of FCV, dating from 1970 to 1989, were analysed by western blotting and immunoprecipitation. Varying reactivity to a panel of F9 specific monoclonal antibodies (MAbs) was observed. DNA sequencing of RT-PCR generated clones supported the observation of variation between capsid proteins. Predicted amino acid sequences varied by 11 to 17.5% across the whole capsid when compared to the published F9 sequence. Differences in amino acid sequence were most apparent in previously described hypervariable regions (C and E). Within hypervariable region E differences of 22 to 34% were observed compared to F9. The observed lack of reactivity to F9 MAbs correlated with amino acid changes within previously characterized binding sites within region E.

Nucleotide sequence of UK and Australian isolates of feline calicivirus (FCV) and phylogenetic analysis of FCVs.

We have determined the first complete genome sequence and capsid gene sequences of feline calicivirus (FCV) isolates from the UK and Australia. These were compared with other previously published sequences. The viruses used in the comparisons were isolated between 1957 and 1995 from various geographical locations and obtained from cats showing a range of clinical signs. Despite these diverse origins, comparisons between all strains showed a similar degree of sequence variation within both ORF1 (non-structural polyprotein) and ORF2 (major capsid protein) (amino acid distances of 7.7-13.0% and 8.8-18.6%, respectively). In contrast, ORF3 (putative minor structural protein) sequences indicated a more heterogenous distribution of FCV relatedness (amino acid distances of 1.9-17.9%). Phylogenetic analysis suggested that, unlike some other caliciviruses, FCV isolates within the current data set fall into one diverse genogroup. Within this group, there was an overall lack of geographic or temporal clustering which may be related to the epidemiology of FCV infection in cats. Analysis of regions of variability in the genome has shown that, as well as the previously identified variable regions in ORF2, similar domains exist within ORFs 1 and 3 also, although to a lesser extent. In ORF1, these variable domains largely fall between the putative non-structural protein functional domains.

Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is biologically active.

The respiratory syncytial virus (RSV) G glycoprotein mediates attachment of RSV to cells via an unknown receptor. To study G glycoprotein function we have cloned two variants of the RSV G gene into a Semliki Forest virus (SFV) expression vector, a full length (rG) and soluble (srG) G glycoprotein variant. By immunofluorescence microscopy, rG was found to be predominantly membrane associated, while srG was mostly cytoplasmic. The rG (80-85 kDa) and srG (75-80 kDa) constructs produced heavily glycosylated proteins, however they were slightly smaller than the G glycoprotein expressed in RSV infected HEp-2 cells (85-90 kDa). The biological activity of purified srG was tested by its ability to bind to RSV permissive cells. Purified srG bound to HEp-2 cells and the amount bound increased linearly with the quantity added. Binding was not saturable with the small quantities of protein available. Binding of srG to HEp-2 cells was inhibited (67-68%) by MAb 30 and neutralising anti-G MAb 29. Nonpermissive SF9 insect cells bound 20-50 times less srG than HEp-2 cells. SFV expressed recombinant RSV G glycoprotein should be useful for studying interactions between the RSV G glycoprotein and cells.

Association between the sigma C protein of avian reovirus and virus-induced fusion of cells.

Monoclonal antibodies (MAbs) against a 39 kDa (sigma C) protein of the avian reovirus RAM-1 strain inhibited virus-induced fusion of cells and the protein was expressed on the surface of infected cells. The fusion-inhibiting activity of the three MAbs reacting with the sigma C protein suggest two putative epitopes were involved: one epitope recognised by antibody 6H1 and involved in fusion of both Vero and CK cells and a second epitope recognised by antibody 1G1 involved in fusion of Vero cells but not CK cells. The activity of the MAb 6E2 was intermediate, suggesting it may have been located in an intermediate position between the two putative epitopes and inhibited fusion by steric hindrance.

Rubella virus replication complexes are virus-modified lysosomes.

Replication complexes are membrane-bound cytoplasmic vacuoles involved in rubella virus (RV) replication. These structures can be identified by their characteristic morphology at the electron microscopy (EM) level and by their association with double-stranded (ds) RNA in immunogold labeling EM studies. Although these virus-induced structures bear some resemblance to lysosomes, their exact nature and origin are unknown. In this study, the localization of two lysosomal markers, lysosomal-associated membrane protein (Lamp-1) and acid phosphatase, relative to the replication complexes was examined by light and electron microscopy. Confocal microscopy using antibodies to dsRNA and Lamp-1 showed colocalization of these two markers in the cytoplasm of RV-infected cells. Immunogold labeling EM studies using antibodies to Lamp-1 confirmed that Lamp-1 was associated with RV replication complexes. EM histochemical studies demonstrated the presence of acid phosphatase in the vacuoles of RV replication complexes. Taken together, these studies show that RV replication complexes are virus-modified lysosomes.

Immune response to avian reovirus in chickens and protection against experimental infection.

OBJECTIVES: To assess the efficacy of the vaccination procedure and the effect of the transfer of maternal antibodies to progeny chickens on reovirus pathogenicity. DESIGN: To vaccinate chickens and challenge progeny chickens with high doses of homologous and heterologous viruses. PROCEDURE: High doses of reovirus strains RAM-1, 1091 and 724 were used to induce tenosynovitis lesions. High doses were produced by concentration of viruses grown in cell culture. Then similar doses of viruses were used to challenge immunised chickens progeny. RESULT: Vaccination of breeding hens with the RAM-1 strain of avian reovirus, which resulted in the passive transfer of neutralising antibody to progeny chickens, completely prevented the development of tenosynovitis in 80% of progeny chickens infected with the homologous virus. Even though multiple injections of hens resulted in broadening of the normal type-specificity of the neutralising antibody response against heterologous serotypes of avian reovirus, only marginal protection against strains of two heterologous serotypes of avian reovirus was obtained. CONCLUSIONS: A model for assessing the efficacy of vaccination against avian reovirus strains on clinical sign such as tenosynovitis was developed that overcome the normal low virulence of Australian strains of avian reovirus. Breeding hens can be immunised with Australian strain of avian reovirus with passive transfer of antibody via the yolk to the progeny chickens. Although the neutralising antibody response to three injections of inactivated virus decreased the specificity of the neutralising antibody response against antigenically heterologous strains of avian reovirus, the protective immunity appeared to retain type-specificity.

High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon.

The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.

Feline calicivirus strain differentiation using monoclonal antibody analysis in an enzyme-linked immuno-flow-assay.

Six monoclonal antibodies raised against feline calicivirus (FCV) strain F9 were used in an enzyme-linked immuno-flow-assay (ELIFA) to analyse 55 isolates of FCV. Forty seven field isolates were obtained from cats with acute oral/respiratory disease, chronic oral lesions, and from cats showing vaccine reactions, i.e. clinical signs of FCV infection shortly after vaccination. Eight reference strains including F9 and three vaccine strains based on F9 were also examined. All of the strains of F9, derived from various sources, reacted with all six of the monoclonal antibodies, whereas some of the field isolates did not react with any. In general, the field isolates showed a spectrum of reactivities and selected isolates could be distinguished. However, there were no clear cut differences between the clinical groups. Overall, the oral/respiratory group showed less reactivity with the monoclonals, suggesting they were less related to F9. Although the other groups appeared to be more closely related to F9, none of the isolates tested reacted with all six monoclonal antibodies.

Human immunodeficiency virus type 1 replication is blocked prior to reverse transcription and integration in freshly isolated peripheral blood monocytes.

Peripheral blood monocytes are resistant to productive human immunodeficiency virus type 1 (HIV-1) infection in vitro immediately after isolation. No viral cDNA (either early or late transcripts) was detected by PCR in monocytes exposed to virus on the day of isolation. In contrast, in monocytes cultured for as little as 1 day, initiated and completed reverse transcripts were readily detectable within 24 h of infection with both HIV-1(Ba-L) and primary isolates. The levels of initiated, partially completed, and completed viral DNA copies found 24 h after infection increased progressively with time in culture before infection. Unlike quiescent T lymphocytes, there appeared to be no block or delay in the integration of viral DNA into the genome of susceptible cultured monocytes. With an Alu-PCR method designed to specifically detect proviral DNA being used, integration events were found within 24 h of infection in monocytes cultured for a day or more after isolation. No integration signal was found in freshly isolated monocytes up to 7 days following exposure to the virus. Cloning and sequencing of Alu-PCR-amplified DNA confirmed integration in HIV-1-infected cultured monocytes. Our finding that in vitro replication of HIV-1 is clearly blocked prior to the initiation of reverse transcription in freshly isolated peripheral blood monocytes suggests that these cells may not be susceptible to infection in vivo. Further studies to clarify this possibility and the nature of the block to infection should provide useful information for treatment strategies against HIV-1.

Type-specific antigenicity of avian reoviruses.

The type-specificity of the neutralizing activity in chicken antiserum to avian reoviruses was affected by the method of antiserum production. The neutralizing activity produced in response to virus infection had higher type-specificity than that produced by immunization with inactivated virus emulsified in adjuvant. By using reassortant viruses the induction of type-specific neutralizing activity was shown to be associated with the sigma C (sigmaC) virion protein. Antigenic classification of virus strains based on immunoprecipitation of the sigmaC protein by chicken antiserum was attempted and the results were similar to those obtained by reciprocal serum neutralization tests. One-way immunoprecipitation of the sigmaC protein by antisera to some heterologous viruses, similar to that reported in reciprocal neutralization tests, made it difficult to assign individual viruses to serogroups and showed that the type-specificity of the sigmaC protein was not absolute. The neutralization activity of monoclonal antibodies to the sigmaC protein of the RAM1 strain of avian reovirus suggested there were separate type- and group-specific antigenic domains on the sigmaC protein, and that the group-specific domains may be associated with the induction of antibody against heterologous viruses.