- Volume 71, Issue 8, 1990

Activation of the herpes simplex virus type 2 (HSV-2) large subunit of the ribonucleotide reductase (ICP10) gene by papillomavirus DNA encoding the E2 or E7 proteins was studied directly by immunofluorescence or by chloramphenicol acetyltransferase (CAT) analysis with hybrid ICP10 or IE175 and 38K promoter constructions. Cotransfection with bovine papillomavirus type 1 or human papillomavirus type 16 (HPV-16) E2 DNA enhanced CAT expression from constructions in which CAT is regulated by the ICP10 but not by other HSV promoters. Expression was not enhanced by cotransfection with HPV-16 E7 DNA. Sequence analysis of the ICP10 promoter identified a consensus E2-binding motif. Activation was significantly reduced by site-directed mutagenesis of the consensus motif.

Plasmid DNA including the simian virus 40 (SV40) origin of replication undergoes uncontrolled, runaway replication in cos-1 cells owing to the intracellular production of SV40 large T antigen. Covalent linkage of such plasmids with human papillomavirus type 16 (HPV-16) DNA was found to prevent runaway replication. Replication control was found to be dependent on the presence of HPV-16 DNA sequences including the El open reading frame and part of the non-coding region.

The enhancer of JC virus restricts its gene expression to the brain. In a previous study, we demonstrated an enhancer element, nuclear factor I (NFI) motif, in the middle of the enhancer. Here, we demonstrate that the NFI motif is a tissue-specific element. We further present a new tissue-specific element, SacI motif, just upstream from the NFI motif. These motifs showed transcriptional enhancement both in vitro and in vivo and acted upon a heterologous adenovirus major late promoter. DNase I footprint analyses demonstrated that the SacI motif bound to a brain nuclear factor, and that its binding region overlapped with the NFI motif. Gel shift experiments with the SacI motif revealed that the populations of SacI motif factors in the brain and HeLa cell extracts were different. Together with our previous findings about tissue-specific NFI-like factor(s), cooperation of NFI motif and SacI motif factors may be required for the strong brain specificity of the viral gene expression.

Full-length cDNA clones of the swine vesicular disease virus (SVDV) were constructed from subgenomic cDNA clones in the expression vector pSVL (pSVLSOO). The direct transfection of mammalian cells with plasmid pSVLSOO results in the production of infectious virus. The recovered virus was neutralized completely by anti-SVDV guinea-pig serum, but did show a difference in plaque morphology from the parental virus.

Bluetongue virus tubules were purified from Spodop- tera frugiperda cells infected with a recombinant baculovirus containing the NS 1 gene from bluetongue virus serotype 10, and expressed under control of the Autographa californica nuclear polyhedrosis virus polyhedrin promoter. These tubules were subjected to a variety of chemical and physical treatments and the resulting effects on tubule morphology were examined by electron microscopy. A number of morphological similarities were noted between bluetongue virus tubules and cellular microtubules despite a lack of homology between the component proteins at the primary sequence level. A possible multistranded helical configuration is proposed for the tubule structure.

Cell-to-cell fusion of Aedes albopictus (mosquito) cells infected with dengue and St Louis encephalitis (SLE) flaviviruses was induced by exposure to low pH. The parameters of this low pH-induced fusion were examined. Syncytium formation was maximal in cultures 36 to 48 h post-infection and occurred when cultures were maintained at the acid pH for 15 min at 35 °C. The optimal pH range for fusion was 5·0 to 6·5 for dengue virus-infected cells and 5·0 to 5·5 for SLE virus- infected cells. Syncytia were not observed in vertebrate cells (Vero and BHK) under these conditions despite similar virus yields. Fusion was shown to be ATP- dependent and could be prevented by the addition of either polyclonal antiviral antibodies or monoclonal antibody to the envelope glycoprotein. The lysosomotropic amine ammonium chloride inhibited the replication of SLE virus in both mosquito and vertebrate cells, consistent with the idea that low pH-induced fusion is necessary for virus entry into both types.

A restriction fragment library which covered the entire genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) was constructed using plasmid vectors. By analysis of cloned and viral DNA by double digestion with endonucleases and by hybridization techniques, a complete physical map of BmNPV was constructed for BamHI, Eco RI, Hin dIII, Kpn I, PstI and SmaI. Five regions of repeated sequences containing EcoRI sites were also found and mapped.

The relative susceptibilities and symptom responses of different Brassica species to infection by cauliflower mosaic virus (CaMV) have been compared and related to molecular events of the virus multiplication cycle. Variants of B. rapa (genome descriptor aa) were highly susceptible to infection by CaMV strain Cabb B-JI and contained relatively large amounts of virus; B. oleracea (cc) variants showed low susceptibility and contained small amounts of virus. B. nigra (bb) and allotetraploid species, B. juncea (aabb), B. napus (aacc) and B. carinata (bbcc), showed moderate responses to CaMV. CaMV unencapsidated DNA forms were isolated from different Brassica plants and examined by two-dimensional gel electrophoresis and blot hybridization. Viral RNA was estimated by dot blot analysis. These analyses showed differences in accumulation of key viral replication cycle intermediates within the broad range of host plants studied. The most susceptible species contained relatively small amounts of super-coiled (SC) DNA, a component of the CaMV minichromosome, but abundant viral transcripts and reverse transcription replication products. Tolerant plant hosts contained high levels of SC DNA but low levels of viral transcripts and reverse transcription DNA products. Allotetraploids contained SC DNA, RNA transcripts and replication product levels which were generally intermediate between those of their respective progenitor species. Evidence is presented that accumulation of CaMV SC DNA in the less susceptible host species is probably not due to autonomous DNA replication or tissue-specific expression. We conclude that a major component of the susceptibility of Brassica plants (and probably all CaMV host species) to CaMV infection is the level of viral minichromosome expression, influenced directly by the host genotype.

Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of M r 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (PI90, PI20, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. PI90 is an intermediate cleavage product which is further cleaved to form P60 and PI20. PI20, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

The nucleotide sequences of cloned cDNA copies of satellite and defective interfering (DI) RNAs of cymbidium ringspot virus were determined. DI RNA is 499 nucleotides long and is composed of six stretches of sequence derived from CyRSV genomic RNA. Four of these stretches share common 5′ sequences and the 5′ and 3′ ends are identical to those of genome RNA. Satellite RNA is 621 nucleotides long. In some regions the sequence is very similar (60% to 100% identical) to genomic RNA. A consensus sequence is proposed to be involved in the replication of genomic, DI and satellite RNAs.

A full-length DNA copy of cymbidium ringspot virus RNA was cloned downstream of a phage T7 promoter. In vitro transcripts had no extra nucleotides at the 3′ terminus, and a 5′ end likely to be precisely as in genomic RNA. Transcripts were infective when inoculated into test plants. Northern blots from inoculated plants revealed the presence of genomic and subgenomic RNAs, but not of satellite RNA. Virus particles isolated from infected plants had the same outward aspect and size as those of the wild-type virus and were decorated by an antiserum to CyRSV in immune electron microscopy tests.

DNA complementary to the ninth largest (S9) of the 12 genome segments of rice gall dwarf virus (RGDV) was cloned and its sequence was determined. It is 1202 nucleotides in length and contains one open reading frame which extends for 969 nucleotides from nucleotide 26. It encodes a polypeptide of 323 amino acids with an M r of 35560. The dinucleotide sequence at the 5′ end and the trinucleotide sequence at the 3′ end of the plus strand, 5′ GG—GAU 3′, which are present in the RNA of both wound tumour virus (WTV) and rice dwarf virus (RDV), were also found in RGDV genome segment S9. The nucleotide sequences in the non-coding region at the 5′ terminus and in the 15 nucleotides at the 3′ terminus, which form an imperfect inverted repeat of 10 bp together with the 5′ terminus, are approximately 70% homologous with those of the WTV genome segment S9, but only 30% and 50% homologous with the respective termini of RDV S9.

Mesophyll protoplasts from both susceptible and resistant hosts were inoculated with RNA purified from barley stripe mosaic virus (BSMV) strains CV52 and CV42 using the polyethylene glycol (PEG) method. Protoplasts derived from the susceptible Hordeum vulgare L. cv. Black Hulless were susceptible to both BSMV strains, as indicated by fluorescein isothiocyanate staining and ELISA. More than 80% of protoplasts derived from an oat cultivar resistant to CV52, but not to CV42, were readily infected by either CV52 or CV42. Protoplasts from 10 barley lines resistant to CV42 remained resistant to CV42, although a limited number of protoplasts were infected. Functional resistance in the cultured barley protoplasts, but not in those from oat plants, suggests that resistance in these barley lines may be the result of restriction of replication, whereas resistance in oat plants is more likely due to restriction of cell-to-cell movement.

The nucleotide sequence of a 1355 bp cDNA representing the 3′-terminal sequences of pea seed-borne mosaic virus (PSbMV) was determined. This sequence contained a single long open reading frame (ORF) of 1189 bp ending with a single TAA termination codon. Downstream from the ORF was an untranslatable region of 189 bp followed by eight bp of polyadenylate. The probable location of the PSbMV coat protein codons within the long ORF was determined by comparing the inferred amino acid sequence with other potyviral coat protein sequences and by examining the sequence for a potyviral polyprotein cleavage cassette sequence. Direct chemical sequencing of the PSbMV coat protein revealed it to be blocked at its amino terminus. A partial amino acid sequence representing the N terminus of the protease-resistant core of the coat protein was determined, however. Alignment of the PSbMV coat protein sequence and the sequences of seven other potyviral coat proteins revealed significant homology, ranging from 53.7% for potato virus Y strain D to 43.2% for tobacco vein mottling virus.

A circular, viroid-like satellite RNA (sat RNA) was detected in lucerne transient streak virus (LTSV), from lucerne in South Australia. It was larger than the previously reported sat RNA of LTSV, being similar in size and sequence homology to the 388 nucleotide sat RNA previously shown to be encapsidated by subterranean clover mottle virus (SCMoV) isolated from subterranean clover in Western Australia. This indicates that under field conditions, very similar sat RNAs can be associated with two distantly related sobemoviruses, LTSV and SCMoV. The natural hosts of these viruses are lucerne and subterranean clover, respectively.

The sequence of 1390 nucleotides of the 3′-terminal region of Helenium virus S RNA was determined from cloned cDNA. This portion of the viral RNA contains two major open reading frames (ORFs) encoding proteins of Mr 32·9K and 12·6K. The 32·9K protein, which is similar in size to the capsid protein of the virus, shares marked homology with the coat proteins of potexviruses and two other carlaviruses. The ORF, corresponding to Mr 12·6K, nearest to the 3′-terminal poly(A) tail shows extensive similarity with the corresponding proteins of potato virus S and potato virus M. We propose that this protein may be involved in nucleic acid binding within infected cells.