- Volume 70, Issue 3, 1989

Other workers have reported that vesicular stomatitis virus makes aberrantly long polyadenylic acid [poly(A)] tracts in the presence of S-adenosylhomocysteine (S-Ado-Hcy). In the work reported in this paper, the effects of various analogues of S-adenosylmethionine (S-Ado-Met) and ATP on polyadenylation in an in vitro transcription system were examined to determine whether S-Ado-Hcy exerted its effect on polyadenylation due to its relationship to S-Ado-Met or to ATP. It appeared that compounds which affected polyadenylation were those which were closely related to S-Ado-Met and that had the same L-aminoacyl side chain [(COOH)–CH(NH)2–CH2–CH2–]; the nature of the substituent at the –S+(CH3)– position of S-Ado-Met was less important. These analogues appeared to compete with S-Ado-Met for a binding site(s). These data support a model whereby compounds binding at an S-Ado-Met-binding site may have allosteric effects by causing or preventing conformational changes which are involved in polyadenylation reactions, perhaps by affecting the rate of polyadenylation or of termination.

Molecular cloning was used to express human papillomavirus type 6b (HPV-6b) antigens in Escherichia coli. Seven genomic DNA fragments of HPV-6b which together comprise the complete L1 and L2 open reading frames, known to code for capsid proteins, were cloned and expressed in E. coli as both β-galactosidase and TrpE fusion proteins. Western blots of HPV-6b β-galactosidase fusion proteins using ‘genus-specific’ antisera produced by immunization of rabbits with disrupted bovine papillomavirus type 1 (BPV-1) showed that polypeptides encoded by two DNA fragments from the mid portion of L1 of HPV-6b were cross-reactive. Only one of these two polypeptides reacted with antisera raised against disrupted HPV-1, directly demonstrating that this polypeptide contains the papillomavirus ‘common antigen’. The cross-reactive region was confirmed by reversing antigen and antibody. Polyclonal antisera were raised against the seven HPV-6b β-galactosidase fusion proteins and tested against BPV-1 virion proteins on Western blots. Only antiserum against the mid portion of L1 of HPV-6b reacted with the BPV-1 major capsid protein. HPV-6b fusion proteins were also used to test human sera for antibodies reactive in Western blots. Serum samples from 38 patients with documented HPV-6 infections and from 22 presumably uninfected controls were tested. Antibodies were not detected in any of the sera to any of the seven fusion proteins. HPV-6b β-galactosidase fusion proteins are antigenic and can be used on Western blots to localize immunologically reactive sub-regions of proteins by reacting protein fragments with antisera from immunized animals. However, alternative methods will be required to detect anti-HPV antibodies in human sera.

Seventeen strains of yellow fever virus were compared by immunoblotting using polyclonal and monoclonal antibodies. Strains originating in South America were readily distinguishable from African strains on the basis of differences in the envelope protein. The 17D vaccine strain and strains derived from it differed radically from the parent Asibi and other wild-type strains in that cells infected with 17D did not accumulate intact envelope protein. We suggest that failure to accumulate the major viral surface protein is due to its increased susceptibility to breakdown and that this property may be a contributing factor in the attenuation of the vaccine strains.

Inflammatory cells were isolated from West Nile virus (WNV)-infected CBA/H (H-2k) mouse brains, and their function and cell surface markers were studied. Two populations of cytolytic lymphocytes were detected. One population, which lysed WNV-infected and uninfected L929 (H-2k), YAC-1 (H-2a), P815 (H-2d), OH (H-2KdDk) and 2R (H-2KkDb) target cells, had a phenotype of L3T4– Lyt2– Thy1± asialo GM1+ and hence were natural killer (NK) cells. The other, which lysed WNV-infected cells to a greater extent than uninfected L929 cells, had a phenotype of L3T4– Lyt2+ Thy1+ asialo GM1– and were cytotoxic T cells. In addition, the presence of the latter population was demonstrated by the specific lysis of syngeneic WNV-infected astrocytes, a cell type which is resistant to NK cell lysis. The cell surface markers of isolated inflammatory cells were determined by two colour flow cytometry. This showed that 15 to 40% of the cells were Thy1+, among which about 3% were Lyt2+. No L3T4+ cells were detected.

Stimulation of mouse macrophages with Newcastle disease virus (NDV) leads to a rapid and high interferon (IFN) response. The magnitude of this response is influenced by the mouse genotype. We have analysed NDV-induced IFN production at the protein and mRNA levels in two different populations of macrophages derived from ‘high producer’ C57BL/6 and ‘low producer’ BALB/c mice in vitro. The data indicate that bone marrow and peritoneal macrophages from both strains grown in the presence of L cell conditioned medium (CM) as a source of macrophage colony-stimulating factor 1 (M-CSF) or purified murine M-CSF produce 10- to 50-fold more IFN on a per cell basis than cultures of resident peritoneal macrophages. These differences were also found when steady state levels of IFN mRNA were analysed. Differential analysis for the ratios of IFN-α and IFN-β showed that CM- or M-CSF-cultured macrophages produced equal amounts of both IFN species as determined by specific monoclonal antibodies and hybridization experiments using IFN-α and IFN-β DNA probes, whereas resident peritoneal macrophages induced under identical conditions produced almost exclusively IFN-β. This suggests a stimulating effect of M-CSF on IFN synthesis in NDV-induced cultures of mouse macrophages, which is in part due to additional activation of IFN-α gene expression.

The antibody response in calves to natural infections with bovine respiratory syncytial virus (BRSV) was analysed by radioimmunoprecipitation assays. Antibodies to virus proteins of M r 200K (L), 87K (G), 46K (F1), 41K (N), 35K (P), 28K and 24K (F2), 27K (M), 22K and < 14K could be identified. Recovery of 6- to 7-month-old calves from severe BRSV-associated disease was accompanied by the development of an antibody response to the virus, which was directed mainly to the F and N proteins. Calves of 2 to 3 weeks of age possessed moderate levels of maternal antibodies to BRSV particularly directed to the F and N proteins but became seriously ill after infection. The antibody response in these calves was severely suppressed. In the sera of 4- to 9-month-old calves that died in the course of infection, high antibody levels to the virus were found, which were directed at least to the F and N proteins. The presence or development of antibodies to the F and N proteins appears insufficient for protection against or recovery from BRSV infections.

A combined analysis of hydrophilicity, accessibility and flexibility parameters of the deduced amino acid sequence of measles virus (MV) haemagglutinin (H) was used to select 10 regions for synthesis of 10- or 11-amino acid-long peptides. Nine of these sites are probably exposed on the surface of the protein, as polyclonal sera against either purified MV or purified H bound to these peptides as tested by enzyme immunoassay (EIA). Nevertheless, human sera from acute or chronic MV infection did not bind significantly to any peptide, indicating that the selected sites do not function as natural complete epitopes. All antisera raised in rabbits against keyhole limpet haemocyanin-conjugated peptides had a high titre to the homologous peptide and nine of them bound to MV lysate antigen, purified MV and/or purified H as tested in EIA. None of the sera had haemagglutination-inhibiting antibodies and only one antiserum (against peptide 185–195) had a neutralizing antibody titre of 1/160. Only a minority of the antisera were positive in Western blot (four of 10), radioimmunoprecipitation (two of 10) or immunofluorescence (three of 10). The results indicate that the computer program used in this analysis can predict surface-exposed areas of MV H but that the small peptides synthesized have little resemblance to natural antigenic sites.

A panel of 24 monoclonal antibodies (MAbs) to the G1 or G2 envelope glycoproteins of Hantaan virus were used to determine the surface topography and functional properties of antigenic sites. Nine distinct, partially overlapping antigenic sites, two on G1 and seven on G2, were demonstrated by competitive binding assays. Analyses of the antigenic sites by haemagglutination (HA) inhibition and plaque-reduction neutralization tests showed that all of the sites, except one on Gl, were related to viral HA. Only one of the G1 antigenic sites and two of the G2 sites were involved in virus neutralization. These results suggest that certain epitopes related to HA were not critical for virus neutralization. The nine antigenic sites could be further divided into 13 based upon the serological cross-reactivity of MAbs with viruses representative of each of the four known antigenic groups within the Hantavirus genus of Bunyaviridae, i.e. Hantaan, Seoul, Puumala and Prospect Hill viruses.

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.

Neutralizing monoclonal antibodies raised against type O foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type O convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed.

Neutralizing antigenic domains on bovine coronavirus gp100/E2 were mapped to fragments of this protein by proteolytic cleavage and fragment analysis. The procedure involved analysis of fragments generated after incubation of E2–monoclonal antibody complexes with various proteases. The smallest antibody-bound fragments obtained were a 50K fragment following Staphylococcus aureus V8 protease and submaxillary protease digestion, and a 37K fragment following trypsin digestion. Trypsin also produced a transient antibody-bound 50K fragment. A 40K fragment which was not bound by antibody was observed following digestions with all three proteases. The 50K fragments generated by V8, submaxillary protease and trypsin comigrated on gels and displayed the same altered mobility under non-reducing conditions, suggesting identity of these fragments and indicating the presence of disulphide linkages in these fragments. The 40K fragments generated by these three enzymes also comigrated and displayed the same altered mobility under non-reducing conditions. The 37K trypsin fragment contained both neutralizing domains, A and B.

Three transmissible gastroenteritis (TGE) virus-specific T helper (Th) cell hybridomas have been generated from virus-primed BALB/c mice, by fusion with the thymoma BW5147. The hybridomas responded to purified u.v.-inactivated TGE virus with interleukin production and growth inhibition. TGE virus recognition by the hybridomas was restricted by the major histocompatibility complex: only splenocytes from syngeneic or semi-syngeneic mice were able to recognize the antigen. The three hybridomas were Thy 1.2+, but did not express detectable levels of Lyt 1 or Lyt 2 antigens by fluorescent cell sorting analysis. Only one hybridoma (T. 1J. B5) expressed the L3T4 marker. These hybridomas had helper activity, as they were able to reconstitute in vitro the synthesis of TGE virus-specific antibodies by Th cell-depleted spleen cells from immune BALB/c mice. The antibodies that they induced specifically neutralized by 103- to 104-fold the infectivity of TGE virus, ruling out the possibility of inhibition of virus replication by interferon. These hybridomas could be very useful for identifying antigenic domains in TGE virus recognized by Th cells, which cooperate with B cells in the synthesis of neutralizing antibodies.

A monoclonal antibody (MAb), N2, neutralized human cytomegalovirus (HCMV) infectivity in the absence of complement and recognized a normal cell protein. By immunofluorescence, MAb N2 detected antigens in uninfected human cells, but not in cells of non-human origin. Antigens were present at the membrane and were dispersed diffusely within the cytoplasm. MAb N2 immunoprecipitated a glycoprotein with an M r of 94K from uninfected and infected cells. In infected cells only, it also recognized a protein of M r 34K which was not linked to the 94K M r glycoprotein by disulphide bonds. N2 neutralized both laboratory and field isolates of HCMV. A study of the distribution of the N2 neutralization epitope recognized among fresh isolates of HCMV showed that only 67% of the isolates could be neutralized by this antibody. There was no correlation between the number of in vitro passages and the level of neutralization. The 34K M r polypeptide was present in cells infected by all isolates. It thus appears that, during virus assembly, HCMV acquires a normal cell protein that bears a neutralization epitope.

Human cytomegalovirus (HCMV) infection has previously been associated with the production of immunosuppression. The mechanism by which any such immuno-suppressive effect might be mediated is unclear but previous work has implicated an effect of the virus on monocytes. We have attempted to characterize the immunosuppressive activity produced by in vitro infection of normal monocytes with HCMV strain AD169. We first examined the ability of HCMV AD169 and recent clinical isolates to infect normal peripheral blood mononuclear cells in vitro. We have found by immunofluorescence analysis that only a very limited number of peripheral blood mononuclear cells (0·2 to 0·5%) showed evidence of virus infection as demonstrated by expression of the major immediate early protein. We found that the inhibitory activity of supernatants of monocytes exposed to HCMV which suppressed mitogen-driven T cell responsiveness was associated with a protein of about 95K. Experiments to investigate the mechanism of action of this inhibitor suggested the possibility of mycoplasma contamination and we were subsequently able to isolate Mycoplasma hyorhinis from our AD169 virus stock. When a series of low passage clinical isolates of HCMV were examined for their ability to cause immunosuppression, there was a direct correlation between suppression and the presence of contaminating mycoplasmas. Using mycoplasma-free isolates of HCMV we could demonstrate no immunosuppressive effect on mitogen-mediated T cell proliferation of both unseparated human peripheral blood lymphocytes and nylon wool non-adherent T cells; these virus isolates also did not suppress accessory cell function or interleukin 1 production by monocytes infected in vitro. We conclude that the previously reported immunosuppressive effects of HCMV in vitro may be attributable to the presence of mycoplasmas and are unlikely to be due to expression of HCMV in monocytes. We suggest that mycoplasma contamination of isolates of HCMV may be a more extensive problem than is currently recognized.

The herpes simplex virus type 1 (HSV-1) mutant dl1403 contains a 2 kb deletion within the sequences encoding the immediate early polypeptide Vmw110. Previous experiments showed that although dl1403 exhibits normal patterns of gene expression following infection at an m.o.i. of 5 p.f.u./cell its growth and plaquing efficiency are impaired in low multiplicity infections, particularly in human foetal lung (HFL) cells. We have now investigated the ability of two other human herpesviruses, varicella-zoster virus (VZV) and human cytomegalovirus (HCMV), to compensate for this defect at low m.o.i. in HFL cells. Co-infection with HCMV resulted in greatly increased plaque numbers and the apparent particle/p.f.u. ratios of dl1403 stocks were reduced to values similar to those exhibited by wild-type HSV-1 stocks. Complementation of dl1403 in low multiplicity infections by HCMV and VZV was also demonstrated by an increased yield of the mutant virus and an increase in synthesis of dl1403 DNA. Ultraviolet irradiation of HCMV abolished its ability to complement dl1403 and the presence of adenovirus 5 had no stimulatory effect on dl1403 DNA replication. When HFL monolayers were infected with dilutions of dl403 stocks such that no plaques were produced, replication of the mutant virus could be induced by superinfection with HCMV 7 days after the initial infection. These results indicate that a non-lytic interaction between dl1403 and HFL cells is a more likely consequence of a low multiplicity infection than plaque formation.

The virulence of a deletion variant of herpes simplex virus type 2 (HSV-2) strain HG52 has been determined by intracranial inoculation of 3-week-old BALB/c mice. The variant JH2604 has a 1·5 kb deletion within each copy of the long repeat region (RL) of the genome between 0 to 0·02 and 0·81 to 0·83 map units. JH2604 is avirulent for mice compared to the parental wild-type virus, and fails to replicate in mouse brain in vivo. Correction of the deletion by marker rescue resulted in the isolation of recombinants which gave LD50 values comparable to those of individual plaque stocks of the parental HG52. Introduction of the deletion into wild-type virus resulted in recombinants which on intracranial inoculation of mice were avirulent. The results imply that sequences within the 3 kb terminal portion of RL are required for virulence of HSV-2 strain HG52.

Epstein–Barr virus (EBV) from a human hybrid epithelial cell line (NPC-KT), derived from the fusion of human adenoidal cells and EBV genome-containing primary nasopharyngeal carcinoma cells (NPC) has both transforming and early antigen (EA)-inducing abilities. EBV DNA from NPC-KT cells was partially digested with TaqI and ligated to the cloning vector pJB8 at the ClaI site. This cosmid library encompassed the whole genomic DNA of the virus except for several kb of the terminal fragments. The identification and location of each of the cloned DNA fragments have been defined by hybridization to blots prepared with B95-8 and NPC-KT virion DNA. Defective heterogeneous restriction enzyme fragments of the viral DNA were not identified in any of the cosmid clones nor detected in hybridizations to virion DNA, which indicates that a single virus population derived from the NPC tissue has both transforming and EA-inducing activities.

Epstein–Barr virus (EBV) membrane antigen glycoproteins gp340 and gp220 are encoded by a single gene. We have inserted this gene into a bovine papillomavirus (BPV) vector and expressed gp340/220 in mammalian cells under the control of the mouse metallothionein promoter. The proteins produced were of similar M r, showed similar antigenic specificity and were transported to the same subcellular location as the authentic gp340/220. The inclusion of heavy metal ions in the medium had no effect on the levels of gp340/220, which were approximately the same as those found in standard EBV-transformed lymphoblastoid cell lines, e.g. B95-8. Cells that expressed gp340/220 were selected by several rounds of fluorescence-activated cell sorting, but on passage they rapidly lost the ability to express this glycoprotein. In contrast to this we found that BPV-transformed cells expressing a truncated version of gp340/220 still produced it at significant levels after extended passage.

We constructed a recombinant herpes simplex virus (HSV) containing the transcribed coding and non-coding sequences of HSV-1 strain F glycoprotein B (gB) gene, a γ 1 gene, fused to the promoter-regulatory sequences of the HSV-1 α4 gene and inserted into the thymidine kinase gene of RH1G44, an HSV-1 × HSV-2 recombinant that contains an HSV-2 gB gene at the natural locus. Phenotypic analyses of the insertion mutant, R3145, showed that the αgB gene was transcribed in the presence of cycloheximide but underwent partial conversion to the HSV-2 form. Nucleotide sequencing of the gene indicated that the 5′ crossover occurred between nucleotides 107 and 117 upstream from the translation initiation site and that the 3′ crossover occurred between the sequences specifying amino acids 402 and 412 of the HSV-1 gB. The chimeric protein consisted of an N-terminal 405 to 415 amino acids encoded by the HSV-2 gene and a C-terminal 462 to 472 amino acids encoded by the HSV-1 gene. Comparison of the reactivity of the parental and recombinant gB with type-specific monoclonal antibodies indicated that the chimeric gB lost reactivity with four HSV-1-specific antibodies but gained reactivity with three HSV-2-specific antibodies.