- Volume 77, Issue 8, 1996

Several small, raised lesions from the underside of the tongue of domestic rabbits were isolated, and an extract prepared and tested for the presence of rabbit oral papillomavirus (ROPV). Two weeks after inoculation of this extract into the underside of rabbit tongues, multiple small discrete, grey-white nodules were observed that reached a maximum size of 2 mm in diameter by 5 weeks. These lesions showed typical ROPV pathology, and nuclei stained positive for papillomavirus (PV) group-specific antigen (GSA) by immunocytochemistry. Tissue fragments from rabbit tongues were incubated with a suspension of ROPV and placed subrenally into athymic mice. After 60 days, cysts were removed, sections cut for histology, and a virus stock prepared. GSA staining and in situ hybridization demonstrated that the xenografts were morphologically transformed with areas showing strong nuclear staining for viral capsid antigen and ROPV DNA. Extracts prepared from the pooled xenografts contained infectious ROPV as demonstrated by inoculation into the undersurface of tongues of non-immune New Zealand White rabbits. The results demonstrated that stocks of infectious ROPV can be prepared in the athymic mouse xenograft system for use in studies on the experimental transmission of a mucosal-targeting animal papillomavirus.

We analysed by ELISA a total of 478 human sera for the presence of antibodies to HPV-11 virus-like particles. The sera were obtained from patients with current genital warts (group CO), from males attending the hospital for fertility disorders (group MA), from blood donors (group BD) and from patients hospitalized for reasons unrelated to HPV infections (group HO). Antibody prevalence was higher in male patients of group CO (23.0%) as compared to males of groups MA (3.2%; P < 0.0001), HO (5.3%; P = 0.01) and BD (16.7%; NS). In addition, there was a significant difference in antibody titre between the males of group CO compared to group MA. Within the whole sample the absorbance of sera from females was higher than in specimens from males (P < 0.0001). A small subset of the sera was also tested by radioimmunoprecipitation assay (RIPA). There was good agreement between the data obtained by ELISA and RIPA.

Human papillomavirus type 6b (HPV-6b) is one of the most common causes of human genital warts, an important sexually transmitted disease. Discovery of antiviral therapies for this condition has been hampered by the inability to propagate the virus using standard tissue culture techniques and through difficulties in expressing sufficient recombinant viral proteins in vitro. Replication of papillomavirus DNA requires to viral proteins, E1 and E2. In an effort to establish assays to discover compounds active against this virus, we have co-expressed HPV-6b E1 and E2 proteins in insect cells. We demonstrate that the two proteins form a heteromeric complex which can be purified by sequence-specific DNA affinity chromatography. We also demonstrate that the complex has both E1-associated ATPase and ATP-dependent DNA helicase activity and report further characterization of these functions.

Western blot analysis with monoclonal antibodies, produced in response to immunization with gradient-purified adenovirus 41 (Ad41) virions, identified two epitopes of interest on protein VI of enteric adenoviruses. One epitope is unique to subgenus F adenoviruses (Ad40 and Ad41); the other epitope is common to subgenus A (Ad12, 18 and 31) and subgenus F(Ad40, 41) adenoviruses but is not shared by representative serotypes of subgenera B (Ad3 and 7), C (Ad1, 2 and 5), D (Ad8) or E (Ad4). Alignment of the deduced amino acid sequence of the genes encoding the protein VI precursor (pre-VI) of Ad40 and Ad41 (subgenus F), Ad12 and Ad31 (subgenus A), Ad2 and Ad5 (subgenus C) shows that the N-terminal one-third and C-terminal 23 amino acids of pre-VI are highly conserved. Within the central domain, pre-VI of subgenus F serotypes is more closely related to that of subgenus A serotypes than to pre-VI of the non-enteric subgenus C adenoviruses (Ad2 and Ad5). By expressing random oligonucleotide fragments of the Ad41 protein VI gene as part of a T7 gene 10 fusion protein, the two epitopes of interest were mapped to within the same 14 amino acid region in the central domain of protein VI. Given the association of subgenera A and F adenoviruses with paediatric gastroenteritis, the epitope shared by these serotypes may be functionally significant with respect to gut tropism. In addition, this epitope is potentially valuable as a target for the detection of enteric adenoviruses in clinical specimens.

Adenovirus codes for a protease the activity of which can be regulated in vitro by an 11 residue peptide (GVQSLKRRRCF) derived from another viral protein, pVI. Three cysteine residues, one in the activating peptide and two in the protease (C104 and C122), play a central role in both activation and catalysis. Expression of protease mutants in insect cells has shown that C104 is not essential for proteolytic activity. GVQSLKRRRCF also caused a concentration-dependent increase in tryptophan fluorescence of protease expressed in Escherichia coli that paralleled the increase in proteolytic activity, indicating that activation was accompanied by a conformational change. Tryptophan fluorescence of C104S was not increased by the addition of GVQSLKRRRCF, nor was the fluorescence of wild-type protease increased by the addition of the peptide analogues where cysteine is replaced by aspartic acid or serine, suggesting that C104 is involved in activation and C122 in catalysis.

Three clones of hepatitis B virus (HBV) DNA were propagated from sera of each of five patients with chronic hepatitis B who possessed hepatitis B surface antigen (HBsAg) and antibody to HBsAg in their serum. The clones were sequenced within the envelope gene (the preS1, preS2 regions and the S gene). Clones from four patients had various missense mutations involving codons 124–147 of the S-gene which encode amino acids in the loop structures that form the conformational, common antigenic determinant of HBsAg. Clones from three patients had Asn-130 (Gly in the wild-type), which generated a potential N-glycosylation site, Asn-Thr-Ser, spanning amino acids 130–132 of the S-gene product. In addition, clones from one patient had Arg-145 (Gly in the wild-type), which has been reported in escape mutants of HBV. One of the three clones from another patient had Ser-126 in place of Ile or Thr in wild-type HBV, but the remaining two had no mutations known to affect expression of the common determinant of HBsAg. The remaining patient possessed HBsAg of subtype adr and anti-HBs specific for the w determinant. Clones from this patient did not reveal any mutations which are known to affect the common antigenic determinant of HBsAg.

In the Mediterranean region almost all patients with hepatitis B virus (HBV)-related cirrhosis are anti-HBV e antigen (anti-HBeAg)-positive and carriers of HBeAg-negative virus mutants. The six members of a family who acquired HBV infection were recently studied: two siblings developed cirrhosis with persistence of HBeAg positivity, whereas their parents and two more siblings cleared the virus. The two cirrhotic patients showed homozygosity for HLA class I by phenotype, which is a rare occurrence in the general population, while the other family members were heterozygous for HLA class I. The sequencing analyses of the entire viral DNAs isolated from both cirrhotic patients showed that the two viral genomes were almost identical and no mutation preventing HBeAg synthesis or viral gene expression was present. These findings might suggest that homozygosity for HLA class I molecules might be responsible for an insufficient response to the virus, favouring chronic outcome of the infection and the long-lasting persistence of HBV populations that produce HBeAg.

A site capable of strictly host- and cell type-specific recognition was identified in the preS1 domain of woodchuck hepatitis virus (WHV) through the use of antipeptide antisera generated against the extreme N-terminal fragment of the large virus envelope protein. The crucial determinant of this binding site was mapped to amino acids 10–13. Although a synthetic analogue of the site was highly immunogenic, natural WHV envelope did not display the site activity unless it was modified by proteolysis or acidic pH treatment, indicating an internal location of the determinant in viral envelope. Synthetic peptides encompassing the sequence of this site bound woodchuck lymphoid cells and hepatocytes in a species-restricted manner which followed characteristics of a specific ligand-receptor interaction, although their ability to interact with lymphoid cells was considerably greater than that for hepatocytes. In WHV-infected animals, a netural antibody to the identified cryptic cell-binding site arose independently of that directed against epitopes of unmodified virus envelope and its appearance constituted the earliest immunovirological indicator of virus invasion. Our results demonstrated that the preS1 domain of the large WHV envelope protein is endowed with the species- and cell type-specific recognition site which is protected against antibody surveillance by the natural tertiary structure of the protein and we suggest that proteolytic cleavage is required to induce the binding activity.

The herpes simplex virus type 1 (HSV-1) immediate early protein IE63 acts post-transcriptionally to affect RNA 3′-processing and splicing. Functional domains such as the RGG box and zinc-finger motifs potentially provide the protein with RNA binding capacity. Here, IE63 protein expressed in E. coli, purified by affinity chromatography and used in RNA binding assays, demonstrated similar binding to RNA substrates containing poly(A) sites from different temporal classes of HSV-1 genes, RNA containing splice site recognition sequences and RNA containing no recognized processing motifs. Competition binding assays showed that IE63 binding could be competed out, suggesting that IE63 binds RNA weakly. HSV-1 infection results in an increase or stabilization in vitro of protein binding to poly(A) site-containing RNAs; IE63 is required for this effect. RNA binding assays combining purified IE63 with protein from mock-infected and HSV-1 infected nuclear extracts demonstrated no effect on protein-RNA binding patterns.

A mutant herpes simplex virus type 1, termed ΔTfi, with a 350bp deletion of the Sp1, NF-κB, TAATGARATs, C/EBP and F2 DNA-binding elements from -420 to -70 relative to the transcriptional start site of ICPO (Vmw 110), was generated and characterized. The efficiency of plating of ΔTfi was reduced on Vero cells and it expressed correctly initiated ICPO RNA in the presence of cycloheximide, although RNA levels were 2.5-fold lower than with wild-type (KOS) and marker-rescued (ΔTfiR) viruses. This was consistent with a demonstrated reduction in ICPO protein expression for ΔTfi at early times post-infection and a 3-fold reduction in ICPO-dependent transactivation of the ICP6 promoter. KOS, ΔTfi and ΔTfiR replication in murine corneas and trigeminal ganglia were comparable when measured on a complementing cell line, but ΔTfi titres appeared 15- to 50-fold lower when measured on Vero cells. ΔTfi was correspondingly less virulent than wild-type or marker-rescued viruses in both immunocompetent and SCID mice. ΔTfi, however, established and reactivated from latency with efficiencies comparable to wild-type and marker-rescued viruses. These results demonstrate that although this deletion of the ICPO promoter results in lower levels of ICPO in vitro and decreased virulence in vivo, the establishment of and reactivation from latency are unaffected. This indicates that elements which regulate ICPO expression and virulence during acute infection may be distinct from those involved in reactivation.

We describe the nucleotide sequence, transcription pattern and open reading frames (ORFs) located on BamHI restriction fragment 9 (0.406–0.435 map units) in the unique long segment of the pseudorabies virus (PRV) genome. The fragment contains three nested genes with a common 3′ end. The 5′ ends of the corresponding 0.9, 1.7 and 3.3 kb mRNAs have been mapped. Fragment BamHI-9 contains three complete ORFs, ORF1, ORF2 and ORF2.5. ORF1, which is within the 3.3 kb transcript, encodes a protein with an apparent molecular mass of 60 kDa which is homologous to the product of the herpes simplex virus type 1 UL25 gene. The 1.7 kb mRNA contains ORF2, whose product is homologous to the herpesvirus proteinases, while the 0.9 kb transcript contains ORF2.5, which probably encodes the assembly protein precursor. ORF2 was identified as the PRV proteinase gene following expression in E. coli, using the product of ORF2.5 as the substrate protein.

Polyclonal anti-idiotype antibodies (anti-ids) to anti-bovine herpesvirus 1 (BHV-1) MAbs blocked virus infection in cell cultures, indicating that they contain internal images of a viral attachment protein(s). In the present study anti-id (anti-83) of BHV-1 gB was used as a probe to detect the cellular receptor. Anti-id specifically identified a 56 kDa protein in radioimmunoprecipitation analysis (RIPA) of Madin-Darby bovine kidney (MDBK) cell membranes suggesting the involvement of this cell surface component in BHV-1 binding. Anti-83 pretreated with MAb 83 failed to identify the 56 kDa cellular component proving its specificity for the reacting epitope of MAb 83. The recognition of 56 kDa protein by anti-id was inhibited by prior incubation of radiolabelled membrane proteins with BHV-1 suggesting that the ligands competed for the same binding sites on the cells. 35S-Radiolabelled BHV-1 virions also bound a 56 kDa protein from purified MDBK cell membrane proteins in a virus overlay protein blot assay. RIPA using anti-id as probe detected the 56 kDa protein in permissive MDBK cells but not in non-permissive bat lung cells. The protein nature of the 56 kDa component was confirmed by protease treatment of membranes which resulted in abolition of the 56 kDa signal in RIPA. In addition, purified 56 kDa protein inhibited biotinylated BHV-1 attachment in flow cytometry. These findings indicate that the 56 kDa protein identified by anti-id is a putative receptor for BHV-1.

Epstein—Barr virus (EBV) induces human B cell transformation and is closely associated with naso-pharyngeal carcinoma. The expression of an EBV latent membrane protein, LMP-1, protects B cells from apoptosis by up-regulating the expression of a cellular oncogene, bcl-2. LMP-1 also transforms rodent fibroblasts and affects the differentiation, morphology and growth of human and rodent epithelial cells. In this report, we describe a novel finding that high level expression of the LMP-1 gene in a human epithelial cell line (RHEK-1) induces apoptosis, characterized by chromosomal DNA fragmentation in the transfected cells. In particular, such an effect was more apparent under serum starvation. We also found that in the transfected RHEK-1 cells, LMP-1 expression neither affected bcl-2 expression nor led the cells to grow in semisolid soft agar medium. These results indicate that LMP-1 may participate in the development of EBV-associated epithelial malignancy via a mechanism different from that seen in B cell or fibroblast transformation.

Thymidine kinase (TK) activity was detected following expression of the TK gene of Epstein—Barr virus (EBV) using the pET expression plasmid and E. coli BL21(DE3)pLysS. To study the amino acid residues required at the C terminus of the EBV TK protein for enzymatic activity, a series of C-terminal deletion mutants was generated by direct truncation, linker insertion or PCR mutagenesis to create stop codons at particular sites. Deletion of nine residues from the C terminus caused a 35% reduction in TK activity, while a ten-residue deletion completely abolished the activity. A single point mutation at residue Cys570, corresponding to Cys336 of herpes simplex virus TK, did not alter the TK activity. Single amino acid changes within the last seven to ten residues also did not affect activity. The results indicate that maintenance of the conformation of the C terminus is important for enzyme activity.

Human herpesvirus 7 (HHV-7) is a recently isolated herpesvirus that has been shown to be related to human cytomegalovirus and human herpesvirus 6 and to be a member of the betaherpesvirus subgroup. Here we report the cloning, restriction endonuclease mapping and partial sequence analysis of HHV-7 strain JI DNA. Virus particles were obtained from the supernatant of infected SupT1 cells, the DNA isolated by proteinase K treatment-phenol extraction, and full-length viral DNA was purified and isolated on a pulsed-field gel. Aliquots of this highly purified material were treated in the following ways: (i) sonicated and end-repaired to create short randomly sheared fragments for cloning into M13mp18-Smal vector DNA; (ii) cut with EcoRI for cloning into EcoRI-cut λZAPII or λDASHII vectors; (iii) cut with BamHI for cloning into BamHI-cut λZAP-Express or λDASHII vectors. Partial nucleotide sequencing of the M13 clones followed by detection of open reading frames and their translation allowed the identification of homologues through FASTA searches of the database. Relevant M13 clones were used as probes to isolate corresponding λ phage clones, which could tentatively be mapped to the genome on the basis of presumed genetic collinearity between HHV-7 and HHV-6. Genomic ‘walking’ between EcoRI and BamHI λ genomic libraries enabled overlapping neighbouring clones to be identified and mapped. Each of these clones was analysed to map BamHI, EcoRI, Sall, Smal and Xhol restriction endonuclease sites to provide complete endonuclease maps for the entire genome.

We have performed a systematic search for recombination in the region encoding coat protein and the 3′ non-translated region in natural isolates of potyviruses, the largest group of plant RNA viruses. The presence of recombination, and the localization of the cross-over points, were confirmed statistically, by three different methods. Recombination was detected or suspected in 18 out of 109 potyvirus isolates tested, belonging to four out of eight virus species, and was most prevalent in potato virus Y, clear in bean common mosaic virus, and possible in bean yellow mosaic and zucchini yellow mosaic viruses. Recombination was not detected in the four other potyvirus species tested, including plum pox virus, despite the availability of numerous sequences for this last species. Though it was not specifically researched, no evidence for inter-specific recombination was found. For several reasons, including the fact that only a minor portion of the genome was analysed, the above figures certainly represent an underestimate of the extent of recombination among isolates of potyviruses, which might thus be a common phenomenon.

Multivesicular bodies (MVB) are membranous cytoplasmic inclusions that are invariably associated with tombusvirus infections regardless of the virus species, the host, or the tissue examined. MVB are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. MVB derive from peroxisomes in cells infected by a number of tombusviruses including cymbidium ringspot virus (CymRSV) and from mitochondria in cells infected by another tombusvirus, carnation Italian ringspot virus (CIRV). By using common restriction sites in full-length infectious clones, hybrid clones of these two viruses were constructed. In addition, a mutant of CIRV was prepared in which the protein encoded by the first open reading frame was shortened by 22 amino acids. All mutant transcripts were viable and infected Nicotiana benthamiana plants. Infected leaf tissue samples were collected, processed for thin sectioning, and observed in the electron microscope. The origin of MVB was shown to be under the control of the 5′ region of the viral genome. A sequence as short as about 600 nucleotides in ORF 1 contained the determinants for formation of MVB from peroxisomes or mitochondria.