- Volume 70, Issue 9, 1989

The DNA hypomethylating agent 5-azacytidine greatly increased the reactivation of alphaherpesvirus saimiri-1 (αHVS) from latently infected rabbit dorsal root ganglia, although it inhibited the virus yield and plaque formation efficiency of αHVS in Vero cells. 12-O-Tetradecanoylphorbol 13-acetate(a protein kinase C activator) and sodium n-butyrate both had a stimulating action on replication in Vero cells but did not affect the release of αHVS from latently infected rabbit dorsal root ganglia.

We studied the association of herpes simplex type 1 (HSV-1) glycoprotein D (gD-1) expression in epidermal cells (EC) with virus-specific immunity and protection of mice from fatal HSV-2 challenge. Vaccinia virus recombinants containing gD-1 under the control of an early (VP176) or late (VP254) vaccinia virus promoter were used. Mature gD-1 protein was expressed in VP176-infected EC and they had accessory cell function for HSV-2-induced T cell proliferation of immune lymph node cells (LNC). It was not expressed in VP254-infected EC and they did not act as accessory cells. LNC from VP176- but not VP254-immunized mice proliferated in response to HSV antigen and only VP176-immunized mice had complete long-term protection from HSV-2 challenge.

The Burkitt’s lymphoma-derived cell line Daudi is latently infected with the Epstein-Barr virus (EBV). These cells are very sensitive to the growth inhibitory and differentiation-inducing effects of human interferon-α (IFN) and we have examined the possibility that these responses are due to induction of EBV replication. Our results indicate that only a very small proportion of cells (<0·3%) are induced by IFN treatment to express EBV lytic genes at the RNA or protein level, whereas cell growth inhibition is complete under the same conditions. In contrast, when EBV replication is chemically induced the large increase in lytic gene transcripts, including that of the BZLF1 trans-activator gene, is partially inhibited by concomitant IFN treatment. The increase in viral DNA copy number in chemically induced Daudi cells is also partially inhibited by IFN but no effect of IFN on the level of viral DNA is observed in uninduced cells.

Seven temperature-sensitive (ts) mutants of Abelson murine leukaemia virus (A-MuLV) were isolated on the basis of the temperature dependence of their soft agar colony-forming ability. These seven ts mutants exhibited similar characteristics and were not ts for morphological transformation and autophosphorylation of P120gag–abl protein. The dissociation of the properties of morphology, soft agar colony formation and tyrosine kinase activity might suggest that the v-abl product has more than one primary intracellular target.

Complexes of potato spindle tuber viroid (PSTV) with plant nuclear proteins were investigated by in vitro reconstitution of purified viroids and proteins from nuclear extracts, on nitrocellulose filters and in solution. Well defined complexes were formed under both conditions. Competition for complex formation was achieved by greater than a 1000-fold excess of 5S RNA. Linear PSTV transcripts form complexes with protein that are different from those of circular PSTV. Natural viroid–protein complexes were crosslinked covalently by u.v. radiation in nuclei isolated from infected plant tissue. They have a sedimentation coefficient of approximately 10S, and exhibit defined bands in SDS–PAGE gels which are similar to those of viroid–protein complexes reconstituted in solution. A 43K protein was isolated from the cellular complexes by RNase digestion. Complexes crosslinked by u.v. radiation eluted together with non-crosslinked PSTV RNA in anion-exchange HPLC on Nucleogen columns. Using HPLC of non-irradiated samples a 43K protein eluted at a KCl concentration of 0·61 m, but only in samples from infected material. The relevance of the viroid–protein complexes for viroid function is discussed.

Intact virions of robinia mosaic virus (RoMV) which were inactive in a rabbit reticulocyte in vitro translation system when suspended in 10 mm-phosphate buffer pH 6·0, exhibited an efficient translation activity after treatment with 10 mm-phosphate buffer pH 8·0. The estimated M r values of the translation products of the intact virions in vitro were 130K, 93K, 77K, 60K, 43K, 34K and 31K by SDS–PAGE. In comparison with the treated intact virions, the total viral genomic RNA produced only three polypeptides, of M r approx. 31K, 25K and 22K. Circular dichroism (CD) measurements of the virions showed that the α-helical structure of the viral coat protein subunits might undergo some conformational changes after treatment under mildly alkaline conditions. Fluorescence emission and quenching analysis indicated that the microenvironment of chromophores in the virions suspended in 10 mm-phosphate buffer pH 6·0 might be different from that of the virions in 10 mm-phosphate buffer pH 8·0. Both CD and fluorescence evidence suggested that as a result of conformational changes in the viral coat protein subunits, the fine structure of the virions might be altered after short exposure to the mildly alkaline buffer, although such alteration was undetectable by electron microscopy. The conformational changes of the viral coat protein subunits and consequently the alteration of the fine structure of the virions could facilitate the gene expression and uncoating of RoMV in vitro.

Insertions and deletions have been introduced into an infectious cDNA clone of M RNA of cowpea mosaic virus (CPMV), in the coding regions of the 58K/48K and capsid proteins. Transcripts derived from these mutant clones appeared to be replicated in cowpea protoplasts as detected by immunofluorescent staining and Northern blotting. However in cowpea plants, mutations in either region restricted the replication of the viral RNAs to the inoculated cells and thus prevented a successful systemic infection of the plant. These results indicate that the M RNA-encoded 58K/48K proteins are involved in cell-to-cell transport of CPMV, and that the virus can spread only if the RNA is encapsidated in particles.

The dicistronic genomic RNA 3 of brome mosaic virus (BMV) was used in experiments on site-specific cleavage by RNase H and subsequent religation of large BMV RNA 3 fragments with T4 RNA ligase. BMV RNA 3 was cleaved at the intercistronic poly(A) tract into two fragments: a long (L-BMV 3) 5′-terminal fragment (M r 0·40 × 106) containing the 3a gene, and a short (Sh-BMV 3) fragment (M r 0·28 × 106) containing the coat protein gene and the 3′-terminal tRNA-like tyrosine-accepting structure. Two or three adenylate residues were present at the 5′ end of Sh-BMV 3 and one adenylate at the 3′ end of L-BMV 3. Afterreligation of these RNA fragments BMV RNA 3 was constructed with a deletion including the entire intercistronic poly(A) tract but not the flanking sequences. The religated RNA 3 replicated in wheat plants co-inoculated with BMV RNA 1 and RNA 2. The normal poly(A) tract was restored in progeny BMV RNA 3 during the course of replication.

The negative-stranded genomic RNA of lettuce necrotic yellows virus (LNYV) was isolated and estimated by denaturing agarose gel electrophoresis to consist of about 13000 nucleotides (nt). Hybridization of randomly labelled LNYV RNA to poly(A)+ RNA from infected Nicotiana glutinosa plants revealed the presence of five distinct complementary RNA species (cRNAs) ranging in size from 900 to 6450 nt. Individual recombinant cDNA clones derived from LNYV RNA hybridized to four of these cRNA species. Each cRNA contained unique sequences which together represented about 95% of the viral genome. From their size range and sequence complexity, it is assumed that the cRNAs represent messenger RNAs encoding the five LNYV structural proteins. The mRNA for the nucleocapsid protein is cRNA 3 because a recombinant cDNA expression vector clone containing sequences which hybridize to cRNA 3 produced a polypeptide which was detected with a monoclonal antibody specific for the nucleocapsid protein.

Insertion and deletion mutagenesis of the two virion-sense genes, V1 and V2, of maize streak virus (MSV) prevents symptomatic infections following Agrobacterium-mediated ‘agroinoculation’ of maize seedlings. These genes code for an M r 10900 protein and for coat protein, respectively. Mutants containing insertions or deletions in the coat protein gene, V2, were able to replicate to low levels, producing dsDNA although virion ssDNA was not detected and symptoms were not observed. Hence, unlike the bipartite geminiviruses, MSV requires coat protein to produce symptomatic systemic infection. Mutations in gene V1 which considerably shortened the M r 10900 protein (V1 gene) resulted either in low levels of replication, in which all the DNA forms associated with a wild-type infection were produced, or in no infection, in which case coat protein production may also have been affected. A V1 mutant generated in vivo with 11 of the 14 N-terminal amino acids altered, was viable and produced symptoms typical of a wild-type infection. Infectivity, assessed by replication and symptom expression, was restored by co-inoculating constructs containing single mutations in different open reading frames, thus rescue can occur by trans-complementation of gene products. The experiments showed that the mutations did not affect the nucleotide sequence requirements for replication and that in all cases intermolecular recombination eventually resulted in dominant wild-type virus.

The RNA genome of papaya mosaic virus is 6656 nucleotides long [excluding the poly(A) tail]with six open reading frames (ORFs) more than 200 nucleotides long. The four nearest the 5′ end each overlap with adjacent ORFs and could code for proteins with M r 176307, 26248, 11949 and 7224 (ORFs 1 to 4). The fifth ORF produces the capsid protein of M r 23043 and the sixth ORF, located completely within ORF1, could code for a protein with M r 14113. The translation products of ORFs 1 to 3 show strong similarity with those of other potexviruses but the ORF 4 proteinhas only limited similarity with the other potexvirus ORF 4 proteins of 7K to 11K.

The nucleotide sequences of cDNA clones corresponding to 2569 nucleotides from the 3′ end of cymbidium ringspot tombusvirus (CyRSV) RNA were determined. This region contains three open reading frames giving rise to three predicted protein products, two of which had been identified in previous studies. The 3′ non-coding region is 351 nucleotides long. The amino acid sequence of CyRSV coat protein has striking similarities with that of tomato bushy stunt tombusvirus, particularly in the S domain. No homology was found between the protein encoded by the second largest open reading frame and the corresponding product of other plant viruses.