- Volume 76, Issue 11, 1995

Pseudorabies virus (PRV) early protein 0 (EP0) contains the RING finger domain with homology to the immediate-early (IE) protein ICP0 of herpes simplex virus type 1 (HSV-1). EP0 was detected by indirect immunofluorescence in the nuclei of the cells transfected with EP0 expression plasmid as is the case in cells infected with PRV. In transient expression assays, EP0 trans-activated the PRV IE, thymidine kinase (TK) and glycoprotein X (gX) promoters, indicating that EP0, like ICP0 of HSV-1, is a transactivating protein.

PML is a protein associated with discrete spherical structures within the nucleus of normal cells. A defect in PML expression is observed in acute promyelocytic leukaemia as a consequence of a chromosomal translocation involving the genes encoding PML and the α retinoic acid receptor (RARα). Disruption of PML bodies also occurs during herpes simplex virus infection after the immediate early protein Vmw110 has become associated with PML bodies. In this study, we followed the fate of PML bodies in human fibroblasts during the course of a human cytomegalovirus (CMV) infection. Disruption of PML bodies was observed to be dependent on de novo CMV gene expression and to occur within 4 h post-infection, concomitant with the onset of CMV IE gene expression. Although a transient increase in the number of PML bodies could be observed in some cells, PML exists predominantly as a diffuse nuclear protein during both the early and late stages of CMV infection. Although the function of PML bodies is still uncertain, their disruption may be important for efficient herpes virus replication.

We have characterized the gene encoding the murine cytomegalovirus (MCMV) homologue of the human cytomegalovirus (HCMV) UL100 open reading frame (ORF) that encodes the HCMV glycoprotein M (gM) molecule. It was identified based on its collinearity with MCMV homologues of the HCMV UL99, UL102, UL103 and UL104 ORFs which lie in the HindIII G fragment of the K181 strain of MCMV. Sequencing of a 2.3 kb EcoRI-BamHI subfragment of the EcoRI G fragment adjacent to the EcoRI A fragment revealed the presence of the complete MCMV gM ORF and two incomplete ORFs, which corresponded to homologues of HCMV UL99 and UL102. The MCMV gM ORF consists of 1059 nucleotides and is expressed as a 1.2 kb transcript at late times post-infection. To precisely characterize the gM transcript, the 5′ and 3′ ends were mapped. It was found that the transcript initiates at nucleotides 740 or 745, and that the site of polyadenylation at nucleotide 1961 occurs downstream of the second potential polyadenylation signal located at nucleotide 1934. Based on these findings the MCMV gM is predicted to consist of 353 residues and when compared with HCMV gM has a 47% level of identity. Of great interest is the finding that the MCMV gM amino acid sequence is completely conserved among six isolates of MCMV that had been shown to exhibit considerable variation both in the MCMV glycoprotein B and the immediate-early 1 gene-encoded pp89 molecule. Thus, this glycoprotein appears to be antigenically conserved.

In the process of generating an insertional mutant of herpesvirus of turkeys (HVT) expressing lacZ at the protein kinase (PK) locus, we isolated a recombinant which contained an intact PK gene but the short unique regions US1, US10 and SORF3 had been deleted and replaced by the lacZ cassette. Moreover, the virus contained duplicate copies of gD, gI and gE in an opposite orientation flanking lacZ, US2 and PK which were contiguous. These results are of interest in relation to the flexibility of the short unique segment (Us) and of the inverted repeats flanking Us of the alpha-herpesviruses. The recombinant expressed β-galactosidase and was genetically stable in vitro and in vivo. Chickens inoculated with the virus developed antibodies to HVT antigens and to β-galactosidase but the replication of the recombinant in vivo was impaired in comparison to parental HVT as shown by a reduction in the proportion of infected lymphocytes.

Human papillomavirus type 16 (HPV-16) DNA replicates episomally and requires two virally expressed proteins, E1 and E2. The E1 protein has both helicase and ATPase activities and is absolutely required for viral DNA replication. The E2 protein is a potent transcriptional activator and greatly increases viral DNA replication by colocalizing E1 to the origin of replication. Recently, we characterized a region of the E2 protein essential for the binding to E1. In this study we have analysed in further detail the nature of the association between E1 and E2. Using an extensive set of E2 mutant proteins we have identified two widely separate regions of the E2 protein which are essential for binding to E1. Interestingly, two E2 mutants which fail to bind E1 also fail to activate gene expression, indicating the existence of multifunctional domains on the E2 protein. In addition, cotransfection of E1 with E2 significantly increases E2 transcriptional activity on an heterologous promoter.

The genome of cocksfoot mottle virus (CfMV) is a positive-sense ssRNA molecule of 4082 nucleotides as revealed by sequencing the entire genome. The 5′-untranslated region of the genome is 69 nucleotides and the 3′-untranslated region is 225 nucleotides in length. The coding region contains four open reading frames (ORFs). The organization of CfMV ORFs differs significantly from that of the previously sequenced sobemoviruses southern bean mosaic virus and rice yellow mottle virus. ORF1 encodes a protein having a calculated molecular mass of 12.3 kDa. The function of this protein is unknown. The next ORF codes for the putative VPg and serine protease. The ORF2a product consists of 568 amino acids, with a calculated molecular mass of 60.9 kDa. The replicase of CfMV is translated as part of a polyprotein by –1 ribosomal frameshifting in ORF2a. The calculated molecular mass of the transframe protein is 103.4 kDa. ORF3 encodes the 27.6 kDa coat protein. This has been verified by amino acid sequencing of the CfMV coat protein N terminus. Northern blots of total RNA from CfMV-infected barley leaves reveal the 4.1 kb genomic RNA band and one virus-specific band of 1.2 kb, which may represent a subgenomic RNA for coat protein synthesis.

Transgenic tobacco plants (V123 plants) expressing a set of full-length brome mosaic virus (BMV) genomic RNAs from the cauliflower mosaic virus 35S promoter were produced. The accumulation level of BMV RNAs in V123 plant cells was approximately 1% of that in non-transgenic tobacco protoplasts inoculated with BMV RNAs. The level of BMV RNA in V123 protoplasts did not increase after inoculating the protoplasts with BMV RNAs, whereas V123 protoplasts supported the accumulation of cucumber mosaic virus (CMV) RNAs to a level similar to that in non-transgenic tobacco protoplasts after inoculation with CMV RNA. Such BMV-specific resistance was also observed in protoplasts from V12 plants expressing full-length BMV RNA1 and RNA2, both of which are required and sufficient for BMV RNA replication. On the other hand, protoplasts from M12 plants, expressing truncated BMV RNA1 and RNA2 in which the 3′ 200 nucleotides required for BMV RNA replication were deleted, exhibited weaker resistance to infection with BMV RNA than V12 protoplasts, although the accumulation level of truncated BMV RNA1 and RNA2 in M12 protoplasts was higher than that of BMV RNA1 and RNA2 and V12 protoplasts. These results suggest that expression of BMV RNA replicons is involved in the induction of resistance, rather than high-level accumulation of BMV RNAs and/or their encoded proteins.

The complete genomic sequence of pothos latent virus (PoLV) has been determined. The genome organization is very similar to that of tombusviruses. The genome is 4415 nucleotides long and contains five ORFs. The 5′ ORF (ORF 1) encodes a protein with a predicted molecular mass of 25 kDa and readthrough of its amber stop codon results in an 84 kDa protein (ORF 2). ORF 3 encodes the 40 kDa capsid protein. Two nested ORFs (ORFs 4 and 5) in different reading frames encode 27 and 14 kDa proteins, respectively. Amino acid sequence alignments revealed significant similarities between the readthrough portion of ORF 2 and the coat protein (ORF 3) of PoLV and the corresponding proteins of several tombusviruses. Conversely, the predicted products of ORFs 1, 4 and 5 showed only limited similarity to the equivalent tombusvirus proteins. These results support the conclusion that PoLV is a new but atypical member of the family Tombusviridae.

The genome of cowpea mottle virus (CPMoV) is a positive ssRNA of 4029 nucleotides with six major open reading frames (ORFs). A non-coding region of 34 nucleotides precedes the first AUG. ORF1 encodes a 25 kDa polypeptide of unknown function and ORF2 encodes a 56 kDa putative RNA replicase. Like other members of carmoviruses, suppression of the amber termination codon of ORF1 would produce a read-through polypeptide of 83 kDa. ORF3 and ORF4 encode two small proteins of 7.8 and 9.8 kDa, respectively. ORF5 encodes the 40 kDa capsid protein. ORF6 is located within ORF5 but is in a different frame and has no postulated function. CPMoV RNA is blocked at the 5′ end and is not polyadenylated at the 3′ end. Comparison of the physicochemical properties, genomic arrangement, and predicted amino acid sequences with those of other viruses justify the assignment of CPMoV to the genus Carmovirus, family Tombusviridae.

Tobacco rattle virus (TRV) isolate PPK20 is transmitted by Paratrichodorus pachydermus nematodes. The factor(s) determining vector transmissibility has been shown to be located on TRV RNA 2. Sequence determination revealed that PPK20 RNA 2 contains three open reading frames encoding the coat protein (cp) and proteins with molecular masses of 29.4 and 32.8 kDa. The 29.4 and 32.8 kDa protein-coding genes showed no significant sequence similarity to any other known tobravirus gene. A full-length cDNA of PPK20 RNA 2 cloned between the 35S promoter and nos terminator infected plants when co-inoculated with PPK20 RNA 1. Deletions in the reading frames of the 29.4 and 32.8 kDa proteins revealed that these sequences are dispensable for replication of PPK20 RNA 2 in plants. Subgenomic RNAs for translation of cp and the putative 29.4 and 32.8 kDa proteins were detected in infected leaves. The possible role of PPK20 RNA 2 nonstructural genes in TRV vector transmission is discussed.

The large non-structural polyprotein (206 kDa) of turnip yellow mosaic tymovirus (TYMV) undergoes auto-cleavage, producing N- and C-terminal proteins. Here we show that the viral proteinase responsible for this event is active when produced in Escherichia coli, as monitored in Western blots by examining the generation of the C-terminal cleavage product after induction by IPTG. The outer boundaries and critical amino acids of the proteinase domain were characterized by deletion analysis and site-directed mutagenesis. A miniproteinase of 273 residues resulting from combined N- and C- terminal deletions still performed efficient cleavage. Sequence analysis of the bacterially-purified C-terminal cleavage product indicated that cleavage occurs between Ala1259 and Thr1260 of the non-structural protein.

Algal virus infection proceeds via the specific recognition of the host cell wall, penetration of the cell wall and transfer of genetic material into the cytoplasm of the host cell. This process is similar to that which occurs when bacteriophage infect bacteria so that techniques and concepts developed to study bacteriophage are applicable to algal virus studies. By measuring virus-induced changes in chlorophyll fluorescence we have redefined classical studies on the distribution of infectivity. We show that infectivity does not follow a Poisson distribution with a fixed mean, n. By analysing the infectivity of algal viruses over a broad range of virus:cell ratios we have obtained a corrected Poisson distribution that reflects the probability of multiple virus particles attached per cell and is equally applicable to algal viruses and bacteriophage.