- Volume 73, Issue 5, 1992

A gene designated SalF5R from within the SalI F fragment of vaccinia virus strain WR has been characterized. The predicted primary translation product has 194 amino acids with an M r of 22578 and has features typical of a class I membrane glycoprotein. At the amino and carboxy termini there are runs of hydrophobic residues that might function as membrane signal and anchor sequences, respectively, and after the C-terminal hydrophobic sequence there is a short charged sequence that may prevent passage of the molecule through the membrane. Between these hydrophobic regions there are two potential sites for addition of N-linked carbohydrate. Northern blotting using a probe to an internal region of the open reading frame (ORF) detected an early transcript of 750 nucleotides and late transcripts of heterogeneous length. However, accurate mapping of the 5′ ends of these transcripts did not reveal a monocistronic mRNA that might allow translation of the full-length SalF5R ORF. Instead two early transcripts were found, one beginning 130 nucleotides downstream of the start of the ORF, and a second initiating in the short intergenic region between SalF5R and SalF6R. The nearest late initiation site mapped to the beginning of the upstream gene SalF4R (profilin) and not the beginning of the SalF5R ORF. A virus deletion mutant lacking an internal 554 nucleotides of the 582 bp SalF5R ORF was constructed by insertion of the Escherichia coli guanine phosphoribosyl transferase gene linked to the vaccinia virus p7.5K promoter. The rate of replication and the final titre of intracellular naked virus produced by this virus was indistinguishable from wild-type virus in CV-1 and RK13 cells. In intranasally infected mice the virus lacking SalF5R was not attenuated in comparison to wild-type virus. The SalF5R site may be useful for insertion of foreign DNA into recombinant vaccinia viruses.

The incubation of vaccinia virus cores in appropriate conditions promotes the release of core proteins into a supernatant fraction. Under transcription assay conditions core mRNAs are extruded in association with viral core proteins, however the presence of these proteins within the core particle is not essential for RNA synthesis and extrusion. The RNA-protein complex is resistant to micrococcal nuclease. Five proteins of 60K, 43K, 28K, 18K and 14.5K with RNA-binding abilities have been identified by [32P]RNA overlay protein blot assays. These proteins are likely to be a component of the viral ribonucleoprotein complex since core basic proteins with similar M rs have been identified and at least one RNA-binding protein is predicted in the vaccinia virus genome.

The promoter region of a keratin 6 (K6) gene was used to regulate expression of the early region of human papillomavirus type 1 (HPV-le) in transgenic mice. In one line of mice the K6-HPVle transgene was transcribed in several regions of the skin, the predominant transcript being a 1.1 kb RNA including the E4 open reading frame, and E1–E4 protein was detected in the upper suprabasal layers of the skin in paws and tail. A 1.7 kb RNA corresponding to the E6/E7 transcript was also prominent in tails of homozygous transgenic animals. In young homozygous transgenic mice the epidermis of the tail showed dysplasia and hyperplasia of the suprabasal layers with both hyperkeratosis and focal parakeratosis in the stratum corneum. A similar though milder phenotype was also observed sporadically in hemizygous transgenics. Analysis of the pattern of mouse keratins present in the affected tail skin showed strong up-regulation of the endogenous keratins 6 and 16 throughout the basal and suprabasal layers, suggesting a positive feedback mechanism for the strong transgene activation. Expression of the major differentiation-specific keratins 1 and 10 was repressed. The pattern of E1–E4 expression and the perturbation of normal epithelial differentiation parallel many of the characteristics of HPV-1 warts or verrucae, suggesting that HPV transgenic mice could be useful for analysis of the interactions of HPV gene products with cellular regulatory pathways within an otherwise normal epithelium.

A selection of human sera were tested for the presence of antibodies that neutralized infectious human papillomavirus (HPV) type 11. Neutralizing antibodies were detected by prevention of HPV-11-induced condylomatous transformation of human foreskin chips transplanted subrenally into athymic mice. Test sera were obtained from 21 female patients with genital condylomas and eight patients with laryngeal papillomas. Control patients consisted of 57 adult random blood donors and five asymptomatic children. ELISAs demonstrated that all sera from patients with genital papillomas were strongly reactive to disrupted papillomavirus (PV) antigens of HPV-11, bovine PV type 1 and cottontail rabbit PV, but only two were weakly reactive to intact HPV-11. None of the eight sera from the laryngeal papilloma bearers reacted significantly to disrupted PV antigens, but four of the eight showed strong specific responses to intact HPV-11 only. The majority of the sera that were reactive to intact HPV-11 by ELISA neutralized HPV-11 infectivity in the athymic mouse xenograft system. The data indicated that ELISA reactivity to intact HPV-11 virions was a good predictor for the presence of HPV-11 neutralizing antibodies.

The response of murine T cells to the E7 molecule of human papillomavirus type 16 (HPV-16) was studied using eight different mouse strains of six distinct H-2 haplotypes. HPV-16 E7 protein was prepared as a fusion protein with glutathione-S-transferase, purified by affinity chromatography and used for immunization. Cells from the lymph nodes were cultured with whole fusion protein, glutathione-S-transferase or HPV-16 E7 protein synthetic peptides. All the mouse strains tested, with the exception of BALB/c, recognized the E7 molecule, as evidenced by a proliferative response to at least two of the peptides. The profile of responses to peptides varied between and within a strain, but five distinct immunodominant regions could be identified. These regions were defined on the basis of a reaction to one or more peptides in a given part of the E7 molecule by at least four strains. The five regions were encompassed by amino acid residues 1 to 9, 17 to 32, 42 to 59, 62 to 77 and 87 to 98. The findings suggest that in an outbred population, such as man, the E7 molecule of HPV-16 would be recognized by a large proportion of the population. However, the poor response of two mouse strains [B10.RIII (71NS) and BALB/c] could also have a corollary in man.

The E7 open reading frames of human papillomavirus type 6 (HPV-6) and HPV-16 encode proteins consisting of 98 amino acids that are quite similar in sequence yet different in electrophoretic mobility. Moreover, these proteins vary strikingly in oncogenicity. To investigate the molecular basis of the differences in structure and function, site-directed mutagenesis was used to exchange non-conserved amino acid residues between the two proteins. The mutated coding regions were expressed as fusion proteins in Escherichia coli and identified by Western blotting. Comparative analysis of the affinity-purified mutated E7 fusion proteins in polyacrylamide slab mini-gels in the presence of SDS and 2-mercaptoethanol revealed altered electrophoretic mobilities. This analysis suggests that the aspartic acid at residue 4 (Asp 4) contributes to the characteristic aberrant migration of the HPV-16 E7 protein in SDS-polyacrylamide gels.

Studies with replication-defective mutants of herpes simplex virus (HSV) have defined the minimum requirements for establishment of latency, but their behaviour may not reflect the course of events following infection by wild-type HSV, in which ability to express viral genes has not been precluded by a genetic lesion. To address this issue we devised a strategy for studying establishment of latency by a virulent strain of HSV, based on the distinctive molecular characteristics of latently infected neurons. By combining in situ hybridization for detection of latency-associated transcripts with immunohistochemical analysis of viral proteins we demonstrate here that antigen-positive and latently infected neurons appear synchronously in spinal ganglia during the earliest stages of acute ganglionic infection. This is consistent with early divergence of the molecular pathways leading to productive and latent infection, supporting and extending the results obtained with viral mutants.

The number of herpes simplex virus (HSV) genome equivalents recovered from latently infected mouse spinal ganglia was compared with the proportion of neurons containing latency-associated transcripts (LATs). Two distinct patterns of HSV persistence were observed, depending on the anatomical location of ganglia with respect to the site of cutaneous inoculation. The location of the bulk of latent viral DNA did not correspond with the highest prevalence of LAT+ neurons. Viral DNA was most abundant in spinal ganglia directly innervating the inoculation site and the amount recovered, which was similar to that found previously in human trigeminal ganglia, suggested that LAT+ neurons each contain hundreds of copies of HSV DNA. In stark contrast, although LAT+ neurons were most abundant in neighbouring ganglia, viral DNA was scarce (approx. 20 copies/LAT+ cell). These data indicate that amplification of HSV DNA sequences is greatest in ganglia previously shown to be associated with viral antigen expression during the productive phase of primary infection.

Three major bovine herpesvirus type 4 (BHV-4) glycoproteins have been described previously. By using monoclonal antibodies produced against BHV-4 envelope proteins from which the three major antigens had been removed by immunoaffinity, a fourth glycoprotein was identified. This protein (gp1) has a high M r (> 300K), is detected about 8 h post-inoculation of infected cells and is strictly expressed as a gamma protein. Moreover, gp1 was identified by a polyclonal antiserum from an infected animal, indicating that this glycoprotein is an antigen recognized by the immune system of infected animals.

The infectious genome of a Kenyan isolate of Panicum streak virus (PSV) has been cloned and sequenced. Infection of host plants was done using an Agrobacterium binary vector containing a partial repeat of the genome. Progeny virus from resultant infections proved to be transmissible by the leafhopper Cicadulina mbila (Naude). Comparisons of the amino acid sequences of PSV DNA-encoded proteins with those of previously characterized geminiviruses infecting monocotyledonous plants, including maize streak virus, revealed high levels of identity. The evolutionary relationship between PSV and other geminiviruses infecting monocotyledons is discussed.

The dsDNA of rice tungro bacilliform virus (RTBV) has two discontinuities, one on each strand, each in a specific position as found in other pararetroviruses. The 5′ end of discontinuity 1 was mapped to nucleotide 1 of the published RTBV DNA sequence which suggests that tRNAMet i serves as a primer for negative strand DNA synthesis. This 5′ terminus contains up to two ribonucleotides and the 3′ terminus overlaps it by five to 25 nucleotides. The discontinuity 2 (D2) did not map to a purine-rich region as has been found in other similar viruses. Both the 5′ and 3′ termini of D2 were heterogeneous in position giving structures varying from a gap of 10 nucleotides to an overlap of 103 nucleotides.

The nucleotide sequence of RNA 1 of a German isolate of barley yellow mosaic virus has been determined and compared with a Japanese isolate of the same virus. The sequence identity is 93.6% at the nucleotide level and 96% at the amino acid level. Similar values have been found for the polyproteins of the RNA 2 of both isolates (95%). Both isolates show an RNA 1-encoded protein arrangement similar to that of potyviruses such as tobacco etch virus. In contrast, the polyproteins of the small RNAs (RNA 2) do not show such a similarity to the polyproteins of other potyviruses. However, there is a striking difference between the two isolates in the generally highly conserved active site of the RNA-dependent RNA polymerase. The German isolate exactly matches the consensus sequences for previously described potyviral RNA-dependent RNA polymerases, whereas the Japanese isolate does not.

The complete nucleotide sequence of RNA 4 of rice stripe virus isolate T (RSV-T) was determined and found to consist of 2157 nucleotides, containing two open reading frames (ORFs). One, deduced to be present in the 5′-proximal region of the viral-sense RNA, encodes the stripe disease-specific protein with M r 20541, and the other ORF, in the 5′-proximal region of the viral complementary sense RNA, encodes an unknown protein with M r 32474. Between these two ORFs there is an intergenic non-coding region that could form a secondary structure with two base-paired hairpin configurations. These characteristics indicate that RSV-T RNA 4 has an ambisense coding strategy. Comparison of the two ORFs of RSV-T with those of another isolate revealed 97.2% and 98.0% identity for the nucleotide sequences, and 98.3% and 98.2% identity for the amino acid sequences. The leader sequences of these two isolates were the same. However, an insertion was found in the intergenic non-coding region of RSV-T. Furthermore, comparison of the nucleotide and amino acid sequences of RSV-T RNA 4 with those of RNA 4 of maize stripe virus, which is another member of the tenuivirus group, revealed greater identity, suggesting a close phylogenetic relationship between these two viruses.

Apple stem grooving virus (ASGV) RNA was translated in a rabbit reticulocyte lysate system and shown to direct the synthesis of several polypeptides of M r ranging from 200K to 43K. A polypeptide of 200K was a major product, but no polypeptide with electrophoretic mobility the same as that of the ASGV coat protein was synthesized. Immunoprecipitation experiments showed that a polypeptide of 200K was selectively precipitated by antiserum against purified ASGV. These results indicate that ASGV coat protein is translated as part of a 200K polyprotein.

Double-stranded RNA preparations from Chenopodium quinoa leaves inoculated with two English isolates of beet soil-borne virus (BSBV), BSBV-N and BSBV-452N, a French isolate of beet necrotic yellow vein virus (BNYVV), a Swedish isolate of a tubular beet virus (86–109) or a Belgian isolate of a similar virus (1530) were compared following separation on non-denaturing polyacrylamide gels. The dsRNAs of BNYVV differed in mobility from those isolated from tissue infected with the other four tubular beet viruses, which possessed three major dsRNA species. The degree of sequence identity between BNYVV, BSBV-N, 86–109 and 1530 was investigated by RNA-RNA blot hybridization using 32P-5′ end-labelled probes. Reciprocal hybridization experiments revealed similarity between the BSBV-N, 86–109 and 1530 isolates, but none between these isolates and BNYVV.