- Volume 77, Issue 7, 1996

The glycoproteins of bovine herpesvirus 1 (BHV-1) play important roles in the interactions between virions and target cells. A 108 kDa glycoprotein, designated gII or gp108, has been identified by two different panels of monoclonal antibodies. The gII- and gp108-specific monoclonal antibodies were shown to react with the same protein, which was identified by N-terminal sequencing as the homologue of herpes simplex virus type 1 (HSV-1) gH. When BHV-1 gH was purified by immunoadsorbent chromatography, gL was co-purified. The gH-gL complex induced the production of antibodies that neutralized virus infectivity and inhibited virus penetration. Affinity-purified gH-gL did prevent penetration, but not attachment of BHV-1, which suggests that the gH-gL complex is essential for penetration of BHV-1 into susceptible cells.

Despite the extensive use of antiviral drugs for the treatment of herpesvirus infections and as prodrugs for ablative gene therapy of cancer, little structural information about the drug activating enzyme, herpes simplex virus type 1 thymidine kinase (TK), was available until recently. In the absence of the three-dimensional structure we sought to elucidate the function of the key aspartic acid residue (D162) present within a highly conserved tri-peptide motif that is thought to function in nucleoside binding. In this study we generated a mutant, D162Q, by site-directed mutagenesis, purified both the wild-type and mutant TKs to near homogeneity by single-step affinity chromatography and determined the kinetic parameters for thymidine, ATP, dTMP and dTTP interactions. A 12-fold increase in K m for thymidine by D162Q TK (K m = 6.67 µm) relative to wild-type enzyme (K m = 0.56 µm) was observed and the absence of any alteration in K m for ATP suggests that D162 participates in nucleoside binding. Furthermore, the K i for dTMP is significantly higher for D162Q TK than for HSV-1 TK which is indicative of a shared or overlapping binding site with thymidine. This assessment is further supported by the different inhibition patterns of D162Q and wild-type TKs observed using [α-32P]5-N3dUMP photoaffinity labelling in the presence of thymidine, ganciclovir or dTMP. Interestingly, the K i for dTTP was 30-fold lower for D162Q TK (K i = 2.2 µm) than for the wild-type enzyme (K i = 65.8 µm) which provides further evidence of the importance of D162 in TK function.

The hallmark of Epstein—Barr virus (EBV) infection is the establishment of a viral genome transcription pattern called latency. The EBV BZLF1 gene product EB1 (also known as ZEBRA or Zta) is a transcription factor which is essential for the switch from latency to the lytic cycle. It has been proposed that latency is maintained (i) by the inhibition of EB1 translation via antisense hybridization of EBNA1 and EB1 hnRNAs, or (ii) by the inactivation of the EB1 activating function via the direct interaction of EB1 with ReIA, the retinoic acid receptor and p53, or via the titration of EB1 in RAZ:EB1 inactive heterodimers that are unable to bind to DNA. RAZ, a fusion protein which contains the EB1 C-terminal dimerization and DNA-binding domains fused to the N-terminal 86 amino acids of the EBV BRLF1 gene product R, has been described as a trans-dominant negative regulator of EB1-activated transcription. We demonstrate here that although RAZ efficiently represses EB1-mediated transcriptional activation, the amount of RAZ protein expressed is incompatible with repression through the titration of EB1 in inactive EB1:RAZ heterodimers. Furthermore, we also demonstrate that RAZ efficiently represses transcription activated by an EB1 mutant carrying the GCN4 homodimerization domain (EB1gcn4), despite the inability of RAZ and EB1gcn4 to form stable heterodimers.

A recombinant baculovirus expressing glycoprotein H (gpUL75) of human cytomegalovirus was used to examine the humoral immune response in naturally infected individuals. Recombinant baculovirus infected insect cells produced two forms of gH with molecular masses of 78–82 kDa and 94 kDa. The 94 kDa polypeptide was modified by high mannose oligosaccharide side-chains as shown by reduction in molecular mass after treatment with endoglycosidases H and F. The 78–82 kDa protein represented the non-glycosylated precursor which was resistant to the enzymes. In contrast to gH expressed in mammalian cells, the recombinant baculovirus expressed gH was transported to the cell surface. Glycoprotein H produced in insect cells was reactive with human convalescent sera and all tested neutralizing monoclonal antibodies recognizing either linear or conformational epitopes. Antibodies reacting with insect cell derived gH were detected in 96% of HCMV seropositive human sera. Using insect cells infected with the gH expressing recombinant baculovirus as immunoabsorbent, between 0% and 58% of the total virus neutralizing activity was removed from sera of individuals with a past HCMV infection. gH must therefore be considered a major antigen for the induction of neutralizing antibodies during natural infection.

Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp130 into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp130 remained completely sensitive to endo-β-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.

In all herpesviruses a block of genes is present which is composed of the genes encoding DNA polymerase, glycoprotein B (gB), ICP18.5 and major DNA-binding protein (MDBP). Here we report the cloning and sequencing of this gene block from rat cytomegalovirus (RCMV). The gene block spans 13.3 kbp and contains the four genes in the order pol, gB, ICP18.5 and MDBP. A similar order of genes has previously been reported for human and murine cytomegaloviruses. The pol, gB, ICP18.5 and MDBP genes contain open reading frames which have the capacity to encode proteins of 1120, 914, 893 and 1281 amino acids, respectively. Comparison of the predicted amino acid sequences of the four RCMV proteins with the corresponding proteins of other herpesviruses revealed a close relationship between RCMV and other cytomegaloviruses, which corroborates the usefulness of the RCMV-rat model for studying cytomegalovirus biology.

Degenerate oligonucleotides representing conserved regions of various DNA polymerases hybridized to a region located 26 kb from the left end of the orf virus (OV) strain NZ-2 genome. DNA sequence analysis of this region revealed a 3024 bp open reading frame able to encode a protein with 56% amino acid identity to the DNA polymerase of vaccinia virus (VAC) and with significant homology to other DNA polymerases. Early transcripts derived from the open reading frame were detected in RNA purified from OV-infected cells, and 5′ ends were mapped to a region 8–19 nt downstream from an A/T-rich sequence that resembles VAC early promoters. Unlike the VAC gene, the OV DNA polymerase makes almost exclusive use of G/C coding options. Attempts to substitute the activity of the OV DNA polymerase for its VAC counterpart were unsuccessful. This may indicate that the OV DNA polymerase is incompatible with VAC accessory proteins.

Two myxoma virus transient dominant selection vectors were constructed and used to generate recombinant viruses expressing single and double foreign gene insertions from intergenic sites. The intergenic insertion sites were located between the myxoma virus genes MJ2 (thymidine kinase) and MJ2a, and MA24 (β-subunit RNA polymerase) and MA27 (fusion protein) located approximately 60 and 113 kb from the left-end of the viral genome, respectively. Recombinant myxoma viruses expressing the IacZ gene from either intergenic insertion site retained wild-type virulence. However, expression of the gus gene reduced the virulence of the recombinant viruses in vivo. Northern blot analysis indicated that the major late mRNAs encoding the viral RNA polymerase subunit and fusion protein are both of discrete size. Insertion of a foreign gene under the control of a synthetic late promoter between the MA24 and MA27 genes results in a specific-sized major late transcript for the inserted foreign gene. The MA27 gene transcripts directed by these recombinant viruses are heterogeneous in size, implying the typical pattern of poxvirus late transcription by random 3′-termination prior to polyadenylation. The transcription studies suggest signals located downstream of the insertion site direct 3′-processing of late transcripts irrespective of the gene immediately upstream.

Prophylactic vaccination of cattle with the N terminus (L2a, aa 11–200) of the minor capsid protein L2 completely prevented bovine papillomavirus type 4 (BPV-4) infection of the alimentary canal. To investigate the mechanisms underlying protection from viral infection, sera from vaccinated animals were analysed in neutralization assays both in the nude mouse xenograft system and in cattle. BPV-4 retained its infectivity when incubated with pre-immune cattle sera, whereas, when incubated with immune sera from animals vaccinated with either whole L2 or its N terminus L2a, its infectivity was greatly reduced, indicating that the immune sera had neutralizing activity against the virus. This activity could be abrogated by absorbing the immune sera with L2 or L2a, thus indicating that virus neutralization was due to the presence in the immune sera of anti-L2 antibodies.

The levels of proliferative T cell responses to peptides representing the human papillomavirus type 16 (HPV-16) E7 protein have been measured using short-term T cell lines derived from peripheral blood of healthy women and those with cervical dysplasias and carcinoma of the cervix. In healthy individuals 47% (7/15) responded predominantly to the N- and C-terminal regions of the protein and 6/7 responders were to a single peptide between amino acids 80–94. In comparison 29% (9/31) of women with cervical dysplasia responded to HPV-16 E7, with a significantly reduced response to both the N- and C-terminal regions (P = 0.03 and 0.038, respectively). A higher proportion of responders was found in patients with high grade lesions (56%, 5/9) versus those with atypical or low grade histology (20%, 4/20) and the response to a single peptide between amino acids 75–94 was also increased in this patient group (P = 0.044). This may be a reflection of higher levels of current or previous exposure to HPV-16 in patients with high grade lesions. Correlation of T cell responses with HPV DNA type (detected by PCR of cervical biopsy tissue) showed that 3/9 (33%) HPV-16 DNA-positive individuals responded. This suggests that E7 may not be the dominant target of the immune response or that the response to E7 is down-regulated in these patients. In addition 4/18 (22%) HPV-16 DNA-negative individuals responded, suggesting that their T cells may have been primed by previous exposure to HPV-16 or that a cross-reactive response was detected. Proliferative T cell responses to both HPV-16 E7 and L1 were reduced in women with cervical carcinoma in comparison to those with cervical dysplasia and healthy controls. The observed down-regulation of responses to HPV-16 E7 in women with cervical dysplasia and cervical carcinoma may reflect an altered functional balance between subsets of T helper cells in HPV-16 infections.

Neutralization of human immunodeficiency virus type 1 by adding antibody after the virus has attached to the host cell (post-attachment neutralization: PAN) was investigated using three rat monoclonal antibodies (MAbs) directed against the outer domain of the membrane protein, gp120. Two of the MAbs are specific for the CD4-binding site region and one for the V3 loop. MAb ICR39.13g (CD4-binding site region-specific; IgG2b) effected PAN efficiently at temperatures from 4 to 35 °C. MAb ICR41.1i (V3 loop-specific; IgG2c) effected PAN only at temperatures of 24 °C and below. This suggests that its V3 epitope is masked by a change in gp120 which occurs at temperatures ⩾ 26 °C, or that the virion function which is inhibited by ICR41.1i and is responsible for neutralization has already operated at ⩾ 26 °C. Resistance to neutralization by ICR41.1i occurred within 20 min of shifting the temperature up to 35 °C. Finally, MAb ICR39.3b (CD4-binding site region-specific; IgG2b) did not give PAN at any temperature, indicating that neutralization can only occur if this MAb binds virus before it attaches to the cell. Thus, these studies identify at least one novel fusion-independent event, the neutralization target of a V3 MAb, which occurs very early in the initial stages of virus-cell interaction.

Chinese hamster ovary (CHO) cell lines secreting a series of truncated forms of human immunodeficiency virus type 1 (HIV-1) IIIB (clone BH10) gp120 were assembled. Using purified glyco-proteins, we demonstrated the functional and structural integrity of these truncates by their reactivity with both sCD4 and anti-gp120 monoclonal antibodies (MAbs). Deletion of the V1, V2 and V3 regions had minimal effects on CD4 binding, but deletion of the NH2 terminus affected the folding of the truncated molecule. Deletion of either V1/V2 or V1/V2/V3 regions led to enhanced recognition by some, but not all, MAbs mapping to the CD4 binding site. In contrast, deletion of the V1/V2 regions had no effect on the ability of V3-specific MAbs to bind to the truncate. These results support the use of truncated forms of gp120 as components of potential HIV vaccines.

To elucidate in vivo cell tropism and infection kinetics of an immunodeficiency-inducing isolate of feline leukaemia virus (FeLV-FAIDS), we quantified the two major genotypes comprising FeLV-FAIDS [the replication-competent common form (clone 61E) and the replication-defective variant (clone 61C)] in lymphocyte and leukocyte populations from infected cats. Micromagnetic separation of cell subsets, virus genome-specific PCR and flow cytometry were used to demonstrate the following sequence of events in infected animals: (i) very early replication of both 61E and 61C in CD4 T cells (provirus burden 0.2 to 1 copy/cell at 2–4 weeks post-infection); (ii) lower magnitude replication of both viruses in CD8 T cells and B cells during this initial phase of infection; (iii) plateauing of CD4 cell virus burden accompanied by escalation in CD8 and B cell provirus burdens after 4 weeks; (iv) extensive infection of haemopoietic and circulating myeloid cells. FeLV-FAIDS 61E and 61C replication kinetics and lymphocyte tropisms were similar in blood and lymph nodes, where provirus burdens ranged from 0.15 to 1.0 copy/cell. Moreover, virus infection was productive; 8–48% of blood lymphocytes, 35–81% of node lymphocytes and 53–98% of bone marrow cells expressed FeLV capsid antigen (p27 Gag). These findings suggest that the immunosuppressive potency of FeLV-FAIDS reflects the unique cytopathicity rather than unique cytotropism of its 61C (versus 61E) component.

The genes encoding the inner core protein VP6 of African horsesickness virus (AHSV) serotypes 3 and 6 have been cloned and sequenced. The genes are 1169 nucleotides in length and both encode a largely hydrophilic protein of 369 amino acids. The VP6 amino acid sequence is highly conserved between the two serotypes with an overall similarity of 95%. Comparison of the AHSV VP6 amino acid sequences with those of bluetongue virus serotype 10 VP6 revealed that it is 41 amino acids longer with an overall amino acid identity of 29%. The similarity is mainly confined to a short but highly conserved 13 amino acid region at the N terminus, a short seven amino acid region at the C terminus and a 22 amino acid region close to the C terminus. Within this last region is a smaller 11 amino acid region from 318 to 328 with a 91% similarity to the Rep helicase of Escherichia coli.

Interference of wild-type reovirus growth by some temperature-sensitive (ts) mutant viruses under non-permissive conditions or by other wild-type isolates has been demonstrated; however, the stage of the virus replication cycle at which interference occurs has not been defined. Examination of the time-course of the yields of T1 Lang (T1L) dsRNA in the progeny of mixed infections of T1L with T3 Dearing (T3D) or with a panel of T3D ts mutants at a non-permissive temperature revealed that interference takes place by 8–10 h post-infection and occurs prior to or at the same time as accumulation of reovirus dsRNA. Taken together with our previous results, these data indicate that interference occurs during a window between virus uncoating and synthesis of dsRNA in the reovirus replication cycle, probably at the stage of assembly of primary reovirus particles.

Gnotobiotic (Gn) pigs were orally inoculated with Wa strain (G1P1A[P8]) human rotavirus (Wa HRV) serially passaged in Gn pigs (virulent) or cell culture (attenuated) to determine the median virus infectious dose (ID50) and to assess the site of infection and type and progression of morphological lesions and clinical responses induced by these two strains in Gn pigs. The ID50 of virulent Wa HRV was ⩽ 1 f.f.u. whereas the infectivity of attenuated Wa HRV had to be determined by seroconversion and was ∼ 1.3 × 106 f.f.u. Diarrhoea developed at 13 h post-inoculation (p.i.) in pigs inoculated with ∼ 105 f.f.u. of virulent Wa HRV and correlated with the presence of viral antigen within villous epithelial cells; villous atrophy developed later at 24 h p.i. and correlated with peak faecal viral titres; recovery from disease correlated with the return of morphologically normal villi. Virus, diarrhoea and villous atrophy were not detected in pigs inoculated with ∼ 2 × 108 f.f.u. attenuated Wa HRV although HRV-specific serum antibodies were present by 7 days p.i. These findings demonstrate that virulent Wa HRV infection in Gn pigs occurs primarily within intestinal villous epithelial cells with villous atrophy developing as a sequela to infection. However, factors other than villous atrophy appear to contribute to the early stages of HRV-associated disease expression in Gn pigs. The ability of the attenuated virus to elicit virus-neutralizing serum antibodies without disease or pathology indicates promise in the use of such strains for oral immunization.

The nucleotide sequences of the regions between the membrane and spike protein genes of three strains of porcine haemagglutinating encephalomyelitis virus (HEV) were determined. A total of 739 (HEV strain 67N) and 751 (strains NT9 and VW572) nucleotides were sequenced. Two ORFs, potentially encoding proteins of 12.8 and 9.6 kDa, were identified. Pairwise comparisons with the corresponding ORFs in bovine coronavirus (BCV) and human coronavirus (HCV) OC43 revealed sequence similarities of greater than 88.5% at the nucleotide and 85.3% at the amino acid level for the 12.8 kDa ORF product. For the 9.6 kDa ORF product similarities were greater than 96.9% and 95.2%, respectively. An additional 12 nucleotide deletion upstream of the 12.8 kDa ORF start codon was found in HEV 67N compared to NT9 and VW572. These results reveal a genomic organization of HEV in the region analysed that is homologous to HCV OC43 but different from BCV.