- Volume 79, Issue 1, 1998

Experimental infection of inbred strains of laboratory mice with murine herpesvirus 68 (MHV-68), a natural pathogen of wild rodents, results in acute productive infection of the lung followed by a latent infection of B lymphocytes. We have previously shown that MHV-68 encodes an open reading frame with similarity to poxvirus serpins, designated ORF1, and eight novel tRNA-like sequences. The latter are processed into mature, uncharged tRNAs and are abundantly expressed during both lytic and latent infection. In this study it is demonstrated that deletion of four of the tRNA-like sequences and ORF1 from the virus genome does not affect the ability of MHV-68 to replicate in vitro or to establish, and reactivate from, latency in vivo.

The complete nucleotide sequence of the cDNA genome for garlic virus X (GVX), one of the major viruses infecting garlic plants, was determined. GVX is a single-stranded positive-sense RNA virus consisting of 8106 nucleotides excluding the 3 ′-end poly(A) tail and contains six open reading frames (ORFs) which encode putative proteins of 174 kDa (ORF1), 26 kDa (ORF2), 12 kDa (ORF3), 32 kDa (ORF4), 26 kDa (ORF5) and 15 kDa (ORF6). The putative viral proteins show similarity to those of carlaviruses and potexviruses but show the highest homology to shallot virus X (ShVX). Even though the GVX genome contains most of the structural elements common to carlaviruses and potexviruses, it is distinguished from them by the presence of an ORF4 which encodes an unusual protein. These results suggest that GVX may belong to an unassigned group of ShVX and GarV-type viruses rather than to the carlaviruses or potexviruses.

Mutants of tobacco vein mottling virus (TVMV) with substitutions of Lys or Arg for Asp in the DAG motif at position 5 in the coat protein (CP) failed to infect tobacco plants systemically, but replicated and produced virions in protoplasts. Occasional systemic infections occurred when Nicotiana bentha- miana or transgenic tobacco plantsexpressing wild- type TVMV CP were inoculated with these mutants, but viral progeny contained reversions to negatively or non-charged amino acids at position 5 or substitutions of Glu for Lys at position 8. The compensatory nature of these mutations was demonstrated by recreating one of the most common alterations. Tobacco etch virus (TEV) mutants with substitutions of Lys for Asp in the two DAG motifs near the CP N terminus also failed to infect tobacco plants systemically, and in situ histochemical analysis showed limited movement. A certain net charge evidently must be maintained near the CP N terminus for systemic movement to occur.

Potato plants carrying the Rysto gene from Solanum stoloniferum are extremely resistant to a number of potyviruses, but it is not known at what stage of infection the resistance is expressed. The resistance maybe due to Rysto or to a closely linked gene. In this investigation, we used potato virus Y (PVY) and a tobacco etch virus construct that encodes β-glucuronidase (TEV-GUS) to monitor virus infections of potato plants. Systemic spread of either virus in resistant potato plants was not detectable by serology, RT-PCR, GUS assay or bioassay although each replicated in the initially infected cells of leaves from resistant potato cultivars and was transported into neighbouring cells. However, 3 days post-inoculation (p.i.) a necrotic reaction set in that stopped movement and accumulation of both viruses by 7 days p.i. The resistance reaction (probably a hypersensitive reaction) became visible as necrotic streaks on veins on the lower leaflet surfaces of some potato cultivars carrying the Rysto gene and may be elicited by a common potyviral gene product.

The interactions of seven isolates of peanut stunt cucumovirus (PSV) originating from North America, Europe and Africa, and two variants of PSV satellite RNA (sat RNA) were analysed. Electrophoretic and immunoblot analyses of the coat protein (CP) and Northern blot hybridization analyses of the viral RNAs showed that isolates PSV F352, 1339 and 1507 belonged to subgroup I, and isolates PSV W, Su and B to subgroup II. The seventh isolate, robinia mosaic virus (RoMV) clustered with subgroup I isolates by CP analysis, but was related to both subgroups by RNA hybridization analysis. The ability to support the accumulation of two newly described sat RNA variants, P4 and P6 sat RNAs, was not related to PSV isolate classification: neither PSV W nor RoMV were helper viruses for these PSV sat RNAs. Symptom modulation by both sat RNAs was the same: the presence of sat RNA did not modify the symptoms induced by subgroup I isolates but exacerbated the symptoms induced by subgroup II isolates in both tobacco and cowpea. Sat RNAs P4 and P6 contained 393 nucleotides, and differed only in three nucleotide substitutions. This resulted in marked differences in infectivity, level of accumulation and relative encapsidation between both the sat RNAs. Accumulation levels and relative encapsidation of sat RNAs was also affected by the isolate of helper virus.

The role of the 3′ noncoding (NC) region of Physalis mottle tymovirus genomic RNA in the multiplication of the virus was examined using an in vivo protoplast assay system. Coat protein (CP) synthesis was specifically inhibited by sense 3′ NC region transcripts. To establish the role of the pseudoknot structure present in the NC region in virus multiplication, four site-specific mutants, two of which disrupted the pseudoknot structure while the other two restored the structure, were constructed. Interestingly, none of the four sense mutant transcripts inhibited CP synthesis, suggesting that the specific sequence representing the 3′ terminal pseudoknot structure may play an important role in virus multiplication. However, the wild-type antisense 3′ NC transcript as well as the four antisense mutant transcripts inhibited CP synthesis, suggesting that the inhibitions by antisense transcripts could be due to the formation of RNA-RNA hybrids at the 3′ end of the genomic RNA.

The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5′ ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequi-virus families. The 3′ ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.

Among 14 temperature-sensitive, growth-defective mutants of mycobacteriophage L1 showing a lysis-defective phenotype at 42 °C, six are, in addition, defective in phage DNA synthesis at 42 °C. In the present study, we show that one of the latter six mutants, L1G27ts901, is also defective in the synthesis of both an L1-specific exonuclease (a representative delayed early protein), and of RNA in both the delayed early and late periods but not in the immediate early period. The results of a temperature-shift experiment suggest that the synthesis of L1 exonuclease is regulated by G27 at the level of transcription. Furthermore, the temperature-sensitive defect in delayed early and late RNA synthesis could be largely overcome when the L1G27ts901-infected culture was shifted from 32 to 42 °C at 10 min but not at zero time post-infection. These results suggest that the primary effect of the G27ts901 mutation is to make the phage defective in transcription of delayed early genes at 42 °C, and the defect in late RNA synthesis by this mutant is a secondary effect which is caused by its inability to express regulatory gene products. We conclude that G27 is involved in the positive regulation of expression of the delayed early genes of L1 at the transcriptional level.