- Volume 73, Issue 8, 1992

This study was undertaken to determine whether human papillomavirus (HPV) E6/E7 gene transcription in tonsillar carcinomas is correlated with viral DNA integration. Therefore, tonsillar carcinomas containing HPV-16 (n = 2) and HPV-33 (n = 2) DNA were analysed for the viral physical state and transcription of the E6/E7 region. Southern blot analysis, DNA polymerase chain reaction (PCR) and, eventually, two-dimensional gel electrophoresis revealed indications for the presence of only episomal DNA in the HPV-16-containing biopsies and only integrated DNA in one HPV-33-containing biopsy. The second HPV-33-containing carcinoma, from which one biopsy and two resected tumour specimens were analysed, showed a rather complex physical state profile. The biopsy of this tumour contained only episomal DNA, one resected tumour part contained only integrated DNA and the remaining tumour part contained both integrated and episomal HPV-33 DNA. Independent of the viral physical state, all biopsies and resected tumour parts tested showed the presence of E6/E7 transcripts as determined by RNA PCR. The results indicate that E6/E7 transcripts in tonsillar carcinomas can originate from integrated as well as episomal HPV DNA.

Functional analysis of human papillomavirus type 16 E7 protein by complementation with adenovirus E1A mutants in baby rat kidney cells has shown that the retinoblastoma gene product (RB)-binding region of E7 can substitute in trans for that of E1A. An N-terminal E7 mutant was unable to complement an E1A mutant unable to bind p300, indicating that the two mutants were defective for functionally equivalent activities. E7 proteins with mutations within the RB-binding region were also unable to complement either the non-p300-binding E1A mutant or the N-terminal E7 mutant, suggesting that these mutations affect more than just RB binding.

Local secretory immunity in the vagina may confer a degree of protection against heterosexual transmission of human immunodeficiency virus (HIV). Since the vagina has been shown to respond to local immunization, we have undertaken intravaginal immunization of rats with a 20-mer peptide (amino acid residues 102 to 121) of the HIV-1 envelope glycoprotein (gp120). The peptide was administered in combination with an ‘absorption enhancer’, lysophosphatidyl glycerol (LPG), which has previously been shown to promote the absorption of intravaginally administered peptides, while exerting only mild effects on epithelial membrane integrity. Intravaginal immunization with LPG and the peptide induced serum and vaginal wash IgA and IgG antibody responses which were enhanced in comparison to those after immunization with the peptide alone. Serum antibodies induced by both subcutaneous and intravaginal immunization were able to recognize recombinant HIV-1 gp120. However, the rat antiserum displayed no neutralizing activity against the virus. These results demonstrate that LPG is an effective immunological adjuvant for intravaginally administered peptide antigens. An alternative absorption enhancer, bestatin (BES), was not effective as an immunological adjuvant when administered intravaginally and blocked the adjuvant activity of LPG when BES and LPG were used in combination.

Human papillomavirus (HPV) type 16 transcription was analysed by in situ hybridization using 125I-labelled subgenomic riboprobes, from 26 genital intraepithelial neoplastic (IN) lesions, in formalin-fixed biopsies from 18 different cases. Distinct transcription patterns separable by the presence or absence of late gene transcription were detected. In 12 lesions, late gene expression was absent; HPV transcripts corresponding to the E6 and E7 open reading frame (ORF) were detectable in all basal cells and were usually evenly distributed through all layers of the epithelium. Transcripts corresponding to the E1, E2 and E2/E4 ORFs were present in nine of 12 lesions and displayed a similar distribution. In 14 lesions late gene transcripts were present. E6 and E7 transcripts were detectable basally in all but one lesion. The levels of E7 but not E6 transcripts were markedly increased in the superficial cells of differentiating epithelia, with an identical distribution and at similar levels to those of the E2/E4 transcript. We propose that the most abundant transcript in genital IN lesions containing late gene expression is an E7/E1 ^ E2/E4 transcript corresponding to that reported in HPV-6/11 condylomata and which is derived from a similar promoter within the E7 ORF.

A cDNA fragment encompassing the 5′-terminal half of the NS1 region of the hepatitis C virus (HCV) genome was cloned. The cDNA was expressed in insect cells using a recombinant baculovirus, and a protein band of approximately 21K was identified by immunoblotting with a serum sample from a patient with chronic hepatitis C. Antibody to the protein was detected in sera from 13.4% of patients with chronic non-A, non-B hepatitis (NANBH), 20.8% of patients with liver cirrhosis and 16.8% of patients with hepatocellular carcinoma with no serum markers for hepatitis B virus infection. However, the antibody was not detected in sera from patients with actute NANBH. The prevalence of antibody to the protein encoded by the NS1 region was lower than that of antibody to the HCV core protein, but much higher than that of antibody to the envelope protein. Thus, the NS1 region of the HCV genome is suggested to encode a protein produced during the course of HCV replication.

Supernatant culture fluids from dengue virus type 4 (DEN-4)-infected cultures of monkey kidney Vero cells and Aedes albopictus C6/36 cells contained the virion structural proteins; secreted NS1 was found only in supernatants from infected Vero cells. Using supernatant culture fluids from [35S]methionine-labelled, virus-infected Vero and C6/36 cells, binding of radiolabelled viral proteins was examined with various cell lines varying in susceptibility to DEN-4 infection. Binding of viral E protein was observed with the highly infectible Vero and LLC-MK2 cell lines, whereas a very small degree of binding was seen with four other cell lines (mouse fibroblast L929, bovine kidney MDBK, human hepatoma Hep G2 and primary human endothelial cells) which are less susceptible to DEN-4 infection. The results suggest that cell susceptibility to DEN-4 may be determined largely at the stage of virus binding, i.e. by the presence of a cell receptor capable of binding viral E protein.

Human herpesvirus 6 (HHV-6) induced nuclear antigens in cells as early as 3 h after infection. These nuclear antigens were induced by all three strains of HHV-6 tested, and their de novo synthesis required the function(s) of the intact viral genome. Their appearance was not affected by 2,2′-anhydro(1-β-d-arabinofuranosyl)cytosine, but was completely inhibited by cycloheximide. However, the nuclear antigens did appear if cycloheximide was replaced with actinomycin D. Thus, the nuclear antigens seem to be equivalent to the immediate early antigens of other herpesviruses.

Comparative analysis of DNA sequences located between the coding regions of genes UL49 and UL50 of herpes simplex virus types 1 and 2 (HSV-1 and -2) has revealed a small open reading frame (ORF) of 91 and 87 codons respectively with the characteristics of a genuine protein-coding region. The predicted protein products are clearly related and exhibit features of membrane-inserted proteins, with potential N-proximal signal peptides and C-proximal membrane anchor regions. Counterparts are present in the other sequenced alphaherpesviruses, namely varicella-zoster virus (a previously undescribed gene, 9A) and equine herpesvirus type 1 (gene 10), in the betaherpesvirus human cytomegalovirus (gene UL73) and in the gammaherpesvirus Epstein-Barr virus (gene BLRF1). Therefore, we consider that this ORF represents an additional HSV gene (UL49A) with counterparts in all sequenced alpha-, beta- and gammaherpesviruses.

The complete nucleotide sequence of the genomic RNAs of strains G2 and G7 of soybean mosaic virus were determined. In both cases, the genome is 9588 nucleotides long, excluding the 3′-terminal poly(A) sequence. A large open reading frame (nucleotides 132 to 9329) encodes a polyprotein of 3066 amino acids with a predicted M r of either 349542 (strain G2) or 349741 (strain G7). Based on comparison with the proposed locations of cleavage sites of other potyvirus polyproteins, nine mature proteins are predicted. The mature proteins of the two strains share 94 to 100% amino acid identity, with the greatest variability occurring in the 35K and 42K proteins. Differences in local net charge in portions of these proteins as well as differences in amino acid sequence throughout the genome are discussed in relation to resistance and susceptibility of host plants to strains G2 and G7. Comparison with other potyviruses may be useful for taxonomic clarification of viruses and strains.

The in vitro decapsidation of a small isometric plant virus, turnip yellow mosaic virus (TYMV), was investigated by cryo-electron microscopy. Cryo-electron micrographs of TYMV and empty shells show that rapidly frozen virions still contain their RNA. Images of vitrified virions resemble closely those previously obtained by negative staining. Rapidly frozen virions decapsidate upon thawing although they remain well dispersed on the grid. The escape of the RNA through a hole at the periphery of the capsid could be visualized. The results suggest a model for the in situ decapsidation of small icosahedral viruses.

RNA from the Hungarian isolate of poa semilatent virus (PSLV) directed in vitro synthesis of 120K, 75K, 25K (coat protein) and 20K polypeptides. In vitro translation of PSLV RNA was blocked by the cap analogue, m7Gpp, thus suggesting that the virus RNA was capped. PSLV RNA could be aminoacylated with [14C]tyrosine in vitro. The sequence of 1.5 kb from the 3′ end of the PSLV RNA γ component revealed two open reading frames (ORFs) separated by a uridinerich intergenic region. The putative product of the incomplete 5′-proximal ORF showed a close amino acid sequence similarity with the C-terminal segment of the γa protein (putative RNA replicase) encoded in the barley stripe mosaic virus (BSMV) RNA γ, and the 20K product of the 3′-proximal ORF was found to be related to the 17K γb product of BSMV. The sequence of 0.8 kb from the 3′ end of PSLV RNA β encompassed two (incomplete) overlapping ORFs whose putative products are related to the βc and βd proteins encoded in the similarly arranged ORFs of BSMV RNA β. Nucleotide sequence homology between the respective parts of the two hordeivirus genomes was restricted to the ORF for γa, the spacer between the ORFs for γa and γb, and the 3′ non-coding region, particularly the 95 nucleotide segment at the 3′ end representing a tRNA-like structure. Despite limited sequence conservation beyond this segment, the entire 3′ non-coding region of PSLV RNA could be folded in a tight pseudoknotted structure closely resembling that of BSMV RNA. Surprisingly, the ‘signature’ sequence typical for BSMV RNA, internal polydisperse poly(A) intercalated between the coding part and the 3′ tRNA-like structure, was not detected in the PSLV genome. Instead, the virus RNA contained several oligoadenylate stretches spaced by other residues, close to the junction of its coding and 3′ non-coding portions.

Fragments of the putative non-structural proteins (44K and 46K) encoded by RNA2 of grapevine chrome mosaic nepovirus (GCMV) were expressed as fusion proteins in Escherichia coli and used to raise specific antisera. All three proteins encoded by GCMV RNA2 (viral coat protein, and the 44K and 46K proteins) were detected by immunoblotting in subcellular fractions prepared from the leaves of infected Chenopodium quinoa plants, confirming a previously proposed model of the GCMV RNA2-encoded polyprotein. In addition to the 44K protein, one of the antisera detected a 90K protein presumably representing a precursor of the 44K and 46K proteins. Whereas the 44K and coat proteins could be detected in both soluble and membrane fractions, the 46K protein was found to be specific to the membrane fraction. Analysis of the kinetics of accumulation of the proteins showed that the 44K and 46K proteins were very transient whereas the coat protein was more stable and could be detected up to 21 days after inoculation. These results provide the first direct in vivo data supporting the maturation map of the GCMV RNA2 polyprotein deduced from in vitro experiments.

A procedure based on the polymerase chain reaction (PCR) has been developed to classify cucumber mosaic cucumovirus (CMV) isolates accurately into two subgroups. Two CMV-specific primers that flank the CMV capsid protein gene were used to amplify a DNA fragment of approximately 870 bp. Restriction enzyme analysis of this fragment produces distinct restriction patterns that assign the CMV isolate into one of two subgroups. These two restriction groups correlate with the previously established CMV subgroupings; this PCR-based method may provide a simple alternative to the serological assays used for typing CMV isolates.

Transmissions of virus using the aphid Myzus persicae were performed using plants co-infected with two cucumoviruses, tomato aspermy virus (V-TAV) and cucumber mosaic virus (M-CMV). Five of the aphidtransmitted progeny viruses (3.7%) induced symptoms distinct from those induced by either parental virus. Northern blot hybridization analysis of encapsidated RNAs from these novel progeny demonstrated that all of the RNA profiles were characteristic of pseudorecombinants, i.e. viruses with reassorted genomic RNAs. The two larger RNAs, 1 and 2, originated from V-TAV, whereas RNA 3 was derived from M-CMV. A more sensitive RNase protection assay analysis of both unencapsidated and encapsidated RNAs revealed the presence of minor populations of V-TAV-derived RNA 3 in all of these novel progeny, and of M-CMV-derived RNA 1 (and presumably RNA 2) in one of the progeny. A bias against the encapsidation of the minor populations of RNAs by the M-CMV coat protein was observed, suggesting that there is specificity or competition with regard to the encapsidation of cucumoviral RNAs in vivo. This study demonstrates that insect vectors can mediate the establishment of pseudorecombinants with mixed populations of RNA 3.

We have made transgenic tobacco plants (Nicotiana tabacum, cv. Xanthi nc) expressing the movement protein (P3, 300 amino acids) of alfalfa mosaic virus (AlMV) and two N-terminally deleted proteins lacking respectively 12 and 77 amino acids of the P3 sequence (P3Δ[1–12] and P3Δ[1-77]). The same proteins were expressed in recombinant yeast. By subcellular fractionation, the full-length P3 protein expressed by transgenic plants was found to be associated with cell walls as well as with cytoplasmic particulate material, as was the wild type movement protein expressed by AlMV-infected tobacco plants. P3Δ[1-12] behaved similarly but P3Δ[1-77] was found only in the cytoplasm. It thus appears that a polypeptide domain located between amino acids 13 and 77 of the P3 sequence is necessary for association of the protein with cell walls.

cDNA clones specific for the two genomic RNAs of strain O of the comovirus red clover mottle virus (RCMV) were constructed. Using these clones, in conjunction with clones specific for RNAs of RCMV strain S, local lesion isolates containing reciprocal pseudo-recombinants between strains S and O were identified. Investigation of the biological properties of these pseudo-recombinants showed that the ability of RCMV to infect Chenopodium quinoa is determined by B RNA. The results also suggest that both RNAs are involved in symptom formation in Pisum sativum. Analysis of the strain O clones enabled the sequences at the 3′ ends of both genomic RNAs of strain O to be determined. Comparison of these sequences with the corresponding region of the strain S RNAs suggests that the 3′ terminal sequences critical for replicase recognition may lie somewhat upstream of the poly(A) tract.

Subgenomic messenger RNAs transcribed from the tomato spotted wilt virus (TSWV) S RNA segment were partially purified from total RNA extracts of TSWV-infected Nicotiana rustica and analysed by primer extension analysis. The data obtained show the presence of non-viral sequences, 12 to 20 nucleotides in length, at the 5′ ends of the N and NSs mRNAs, indicating a cap-snatching mechanism for the initiation of transcription. This is the first report of a plant virus using such a mechanism for transcription of the viral genome.

Computer-assisted comparisons of the large proteins involved in the replication of viral RNA have revealed a novel domain located near the N termini of these proteins and conserved throughout the so-called ‘Sindbis-like’ supergroup of positive-strand RNA viruses. This domain encompasses four distinct conserved motifs, with motifs I, II and IV containing an invariant His residue, the AspXXArg signature and an invariant Tyr residue, respectively. Each of the two large groups of viruses within this supergroup, the ‘altovirus’ group (alphaviruses, tobamoviruses, tobraviruses, hordeiviruses, tricornaviruses, furoviruses, hepatitis E virus and probably rubiviruses), and the ‘typovirus’ group (tymoviruses, potexviruses, carlaviruses and apple chlorotic leaf spot virus), can be characterized by additional conserved sequence motifs. Based on the available results of biochemical studies and site-directed mutagenesis of the alphavirus proteins, it is hypothesized that this domain may be involved in methylation of the cap during viral RNA maturation. Unlike the other conserved domains, the RNA-dependent RNA polymerase and the RNA helicase, the motifs typical of the putative methyltransferase domain are universal within the Sindbis-like supergroup but are not found in the proteins of any other viruses, constituting a distinctive hallmark of this supergroup.