- Volume 71, Issue 10, 1990

The genomic sequence encoding a cytomegalovirus strain Colburn homologue (DB129) of the herpes simplex virus major DNA-binding protein (ICP8) was determined. Multiple alignments of the deduced DB129 amino acid sequence and three alpha- and gammaherpesvirus homologues revealed that 56% of the amino acid residues identical in all four homologues are contained within 12 relatively conserved segments, which together constitute only 11 · 2% of the shortest aligned sequence. In light of published ICP8 deletion analyses, this alignment suggests conserved segments that may participate in forming DNA contacts. The identified conserved regions present interesting targets for site-directed mutagenesis in structure-function analyses.

Human papillomavirus type 8 (HPV-8) is one aetiological agent of macular and flat wart-like lesions in patients with epidermodysplasia verruciformis and appears to be closely linked to skin carcinogenesis. A 1.2 kb region of the genome, which was previously shown to contain a viral E2-dependent enhancer, was progressively shortened from both ends with Bal 31. The resulting fragments were tested for their ability to stimulate chloramphenicol acetyltransferase (CAT) expression from the simian virus 40 (SV40) promoter. This analysis showed a complex interaction between cis-active, positive and negative control elements located throughout the non-coding region and the flanking reading frames. Two separate positively acting sequences significantly stimulated expression only in cooperation with a third region, which led to 12-fold, E2-dependent enhancement on its own. A major negative element was not only active in the context of HPV-8 sequences, but also down-regulated SV40 enhancer-promoter-driven CAT expression when cloned downstream of the transcription unit. It acted at the transcriptional level as shown by RNase protection assays and can therefore be regarded as a cis-acting silencer of transcription.

The genomes of Junonia coenia densonucleosis virus (JcDNV) and Galleria mellonella densonucleosis virus (GmDNV) were analysed by restriction endonuclease analysis and Southern blot hybridization. A total of 37 and 33 restriction sites were mapped on JcDNV and GmDNV DNA, respectively. BglI, HaeII and BstEII were site-specific for JcDNV DNA, and BglII and ClaI for GmDNV DNA. The two genomes had nearly identical maps for several restriction endonucleases and Southern blot hybridization using a total genomic JcDNVprobe indicated extensive DNA sequence homologies spanning the entire length of the two genomes. Symmetrical cleavage sites, mapping at the extremities of both genomes, confirmed the presence of inverted terminal repeats of at least 420 to 440 bases in length.

Synthetic peptides P37–49 and P63–79, derived from the pre-S region of duck hepatitis B virus (DHBV), have been synthesized. Only P37–49 was reactive with rabbit anti-DHBs/pre-S antibodies by radioimmuno-precipitation. Antiserum prepared against P37–47 reacted with a 35K polypeptide of native DHBs/pre-S by immunoblotting. It is concluded that P37–49 (MGQHPAKSMDVRR) mimics one of the epitopes of the DHBV pre-S antigen.

This study presents the first nucleotide sequence and deduced primary amino acid sequence of a subtype H1 haemagglutinin from the avian influenza virus A/duck/Alberta/35/76 (H1N1). The molecule is structurally, antigenically and molecularly similar to H1 haemagglutinins of human viruses but sequence homology differences indicate that there has not been a recent transfer of haemagglutinin genetic information between them.

Cell fusion proceeded gradually in measles virus-infected cells incubated at 35 °C. Shift-up of incubation temperature to 39 °C induced rapidly increased cell fusion in spite of the cessation of de novo synthesis of the fusion (F) protein. Pulse-chase experiments showed that there was little difference in the acquisition of immunoreactivity by haemagglutinin (H) and F proteins between the two temperatures. H protein was detected on the cell surface 60 min after the chase at either temperature. However, appearance of F protein on the cell surface took less than 3 h at 39 °C whereas it took 5 h at 35 °C. These data indicate that temperature elevation induces more efficient expression of F protein on the cell surface accompanied by marked syncytium formation in measles virus-infected cells.

Full-length infectious DNA clones were constructed for four distinct phenotypic variants of beet curly top virus (BCTV). Southern hybridization assays indicated that each cloned BCTV genome shared sequence homology with pBCT-028, a full-length infectious DNA clone of a California isolate of BCTV previously characterized by others. Restriction endonuclease maps of the cloned BCTV genomes were distinct from one another. Infectivity assays determined that plasmids containing tandem repeats of BCTV genomes were generally more infectious than excised linear DNA inserts. Progeny virus, derived from plants inoculated with cloned DNAs, differed in their ability to infect sugarbeet, Beta vulgaris L., and the severity of symptoms produced in B. vulgaris and other experimental hosts.

The complete nucleotide sequence of rice dwarf virus (RDV) genome segment 4 was determined. Genome segment 4 was 2468 nucleotides long and had a long open reading frame initiating at nucleotides 64 to 66 and terminating at 2245 to 2247. The deduced polypeptide contained 727 amino acid residues with an M r of 79·8K. Amino acid sequences similar to a ‘zinc- finger’ and purine NTP-binding motifs were present in the deduced polypeptide. Considerable amino acid sequence homology was detected between genome segment 4 of RDV and wound tumor virus (WTV). One of the sequences similar to the ‘zinc-finger’ motif was present in a conserved region of the polypeptide of both viruses. However, the sequence similar to the purine NTP-binding motif was not present in the polypeptide encoded by genome segment 4 of WTV.

The products of partial proteolysis of the M r 126000 in vitro translation products of the RNA of eight tobamoviruses were separated by SDS-polyacrylamide gel electrophoresis. The peptide patterns obtained were compared using a computer program designed to establish phylogenetic relationships. The resulting most-parsimonious phylogenetic trees grouped the tobamoviruses into clusters I (tobacco mosaic virus, tomato mosaic virus, tobacco mild green mosaic virus, pepper mild mottle virus) and II (sunn-hemp mosaic virus, cucumber green mottle mosaic virus, kyuri green mottle mosaic virus), with ribgrass mosaic virus in an intermediate position. This clustering resembles that obtained when the coat proteins of these viruses are compared. If the tobamoviruses have arisen by divergence from an ancestral type, the results suggest that different parts of the genome have diverged similarly and that recombination has not played a major role in the evolution of the group.

By means of monoclonal antibodies (MAbs), five (groups of) epitopes were identified on particles of beet necrotic yellow vein virus (BNYVV). Epitopes 1 and 2, which were located on the opposite extremities of virus particles, are discontinuous (SDS-labile) epitopes which were destroyed when the particles were treated with trypsin. Epitope 3 is a continuous (SDS-stable) epitope lcoated at the same extremity as epitope 2. It was not destroyed when the particles were treated with trypsin and was present on an Escherichia coli- expressed fusion protein containing amino acids (aa) 1 to 103 of the BNYVV coat protein. The continuous epitope 4, which was located along the entire length of the particles, was found to be present on a fusion protein containing aa 104 to 188 of the BNYVV coat protein but not on trypsin-treated virus particles. In Western blots, these treated particles yielded two slightly smaller coat proteins which failed to react with MAbs specific for epitope 4 but did react with polyclonal antisera and MAbs specific for epitope 3. BNYVV coat protein has a trypsin cleavage site on the carboxyl side of arginine in position 182, so it is therefore suggested that epitope 4 is located on the exposed C terminus, which is composed of aa 183 to 188. Epitope 5 was also located along the entire length of the particles but in a more uneven distribution than epitope 4. This may be because it is a discontinuous epitope that is very sensitive to subtle changes in protein conformation.

The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analysed in both rabbit reticulocyte lysate and wheatgerm extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of approximate M r 34·6K was produced. In wheatgerm extracts, four proteins of approximate Mr values 41·6K, 34·6K, 24K and 20K were produced. The genomic locations of the CNV in vitro translation products were determined using several experimental approaches including, first, hybrid-arrested translation using negative-sense RNA corresponding to selected regions of the CNV genome, second, in vitro translation of synthetic positive-sense CNV transcripts and third, in vitro translation of CNV virion RNA fractionated according to size. Together these experiments demonstrated that the protein of Mr 34·6K is derived from the 5′-proximal coding region, the 41·6K protein is derived from an internal coding region, and that at least one but probably both the 24K and 20K proteins are derived from the 3′-terminal coding region. In addition, immunoprecipitation of in vitro translation products using anti-CNV polyclonal serum demonstrated that the 41·6K protein is the coat protein. The templates for the expression of CNV cistrons were investigated by in vitro translation of sucrose gradient-fractionated CNV virion RNA as well as in vitro translation of positive-sense synthetic transcripts.

The nucleotide sequence of RNA 3 of two subgroup I strains of cucumber mosaic virus (CMV), Fny-CMV and M-CMV, was determined and compared at both the nucleic acid and protein level with the previously determined, corresponding (partial) sequences of RNA 3 of five other subgroup I strains: C-CMV, D-CMV, I17F-CMV, O-CMV and Y-CMV. Fny-CMV RNA 3 is composed of 2216 nucleotides (nt) and M-CMV RNA 3 2214 nt. Both RNAs contain two open reading frames, the 3a gene and the coat protein gene. These RNAs showed very little nucleotide sequence divergence, either from each other or from the five other subgroup I strains. The nucleotide sequence variation observed was two to 13 differences in the 120 to 123 nt 5′ non-translated regions, six to 17 differences in the 840 nt 3a genes, two to 15 differences in the 296 to 299 nt intergenic regions, three to 25 differences in the 657 nt coat protein genes and two to 10 differences in the 299 to 303 nt 3′ non-translated regions. Protein sequence similarity was also high, with one to four differences in the 279 amino acids of the 3a proteins and two to 13 differences in the 218 amino acids of the coat proteins. Limited nucleotide sequence variation among nine strains of CMV was also shown using an RNA protection assay and a probe specific for Fny- CMV RNA 3. The limited variation shown by RNA 3 of strains of CMV with different passage histories, isolated in different countries over a 50 year period, suggests that the maintenance of the highly conserved nucleotide sequence may be important for other viral RNA functions or interactions.

An antiserum was raised against a fusion protein containing part of the 56K polypeptide (P5) encoded by the open reading frame (ORF) at the 3′ end of the genome of potato leafroll virus (PLRV). This antiserum reacted specifically with 80K and 90K polypeptides in PLRV-infected protoplasts, with a 90K polypeptide in infected potato tissue and with a 53K polypeptide in protein extracted from purified particles of PLRV. Monoclonal antibodies raised against purified PLRV particles also reacted with these polypeptides, as well as with the 23K coat protein. Virus particles partially purified from infected protoplasts contained some 90K polypeptide as well as the major 23K coat protein. The ORFs of the 23K coat protein and P5 are contiguous and in frame. The results suggest that the P5 polypeptide of PLRV occurs in infected cells as part of a readthrough protein comprising the 23K coat protein joined to the P5 amino acid sequence. Moreover the readthrough protein can be assembled into virus particles as a minor component together with the main 23K component. The P5 protein may thus contribute to properties of PLRV determined by its virus particle surface.

Two ribozymes were synthesized which were designed to cleave potato leafroll virus (PLRV) positive strand RNA within the regions known to encode the viral coat protein and the predicted RNA polymerase gene. DNA sequences encoding the ribozymes were inserted into the Escherichia coli plasmids pTz 18R and pTz 19R under the control of the bacteriophage T7 promoter and enzymically active RNA molecules generated by transcription by T7 RNA polymerase in vitro. Each ribozyme cleaved its cognate site in RNA derived from either cDNA or PLRV particles. Ribozyme cleavage sites within the polymerase gene and coat protein gene were determined and shown to be at the predicted sequence immediately downstream from a GUC motif. An altered version of the ribozyme which recognized the sequence in the coat protein gene was isolated in which a single adenosine residue in the enzymic loop of the ribozyme was deleted. This mutated ribozyme was unable to cleave RNA molecules containing the coat protein ribozyme target site.

Northern blot analysis of Solanum tuberosum infected with potato leafroll luteovirus revealed the 6 kb genomic RNA and a major 2.3 kb subgenomic RNA. The 5′ end of the subgenomic RNA was located at nucleotide 3653 in an intergenic region located at the centre of the viral genome upstream of three open reading frames (ORFs). Transient expression in tobacco and potato protoplasts of the β-glucuronidase reporter gene fused to various putative regulatory sequences present in the subgenomic RNA was used to study their influence on expression levels. We observed a suppression of the amber stop codon separating the coat protein (CP) gene from a downstream ORF (56K protein), to a level of 0.9% to 1.3%. Translation initiation at the AUG of an ORF (17K protein) which is nested within the CP gene, exceeds translation of the CP gene itself by a factor of 7.

The distribution of the helper component (HC) protein of potato virus Y in tissues and cells of infected plants was studied by immunoblotting and immunogold labelling techniques. This HC protein was found in leaf blade and vein tissue but not in the petiole of leaves. In infected cells, the protein was localized in rod-shaped cytoplasmic inclusions known as amorphous inclusions.