- Volume 87, Issue 3, 2006

Kaposi's sarcoma-associated herpesvirus (KSHV) is the latest addition to the long list of human herpesviruses. Reactivation of latent herpesvirus infections is still a mystery. It was demonstrated recently that the phorbol ester TPA was efficient in inducing a reactivation of KSHV infection in the S phase of the cell cycle. In the present study, flow cytometry-sorted, TPA-induced, KSHV-infected haematopoietic cells (BCBL-1) were used to analyse the expression profiles of cancer-related cellular genes in the S phase of the cell cycle compared with the G0/1 phase by using microarrays. Overall, the S phase of the cell cycle seems to provide KSHV with an apt environment for a productive lytic cycle of infection. The apt conditions include cellular signalling that promotes survivability, DNA replication and lipid metabolism, while blocking cell-cycle progression to M phase. Some of the important genes that were overexpressed during the S phase of the cell cycle compared with the G0/1 phase of TPA-induced BCBL-1 cells are v-myb myeloblastosis (MYBL2), protein kinase-membrane associated tyrosine/threonine 1 (PKMYT1), ribonucleotide reductase M1 polypeptide (RRM1) and peroxisome proliferator-activated receptors delta (PPARD). Inhibition of PKMYT1 expression by the use of specific short interfering RNAs significantly lowered the TPA-induced KSHV lytic cycle of infection. The significance of these and other genes in the reactivation of KSHV is discussed in the following report. Taken together, a flow cytometry–microarray-based method to study the cellular conditions critical for the reactivation of KSHV infection is reported here for the first time.

Spodoptera frugiperda cells infected with Autographa californica nucleopolyhedrovirus (AcMNPV) lacking a functional anti-apoptotic p35 protein undergo apoptosis. However, such mutants replicate normally in Trichoplusia ni (TN-368) cells. An AcMNPV plaque isolate (AcdefrT) was identified during propagation of a virus deficient in p35 in TN-368 cells. This virus exhibited enhanced budded-particle formation in TN-368 cells, but was partially defective for polyhedra production in the same cells. Virus replication in AcdefrT-infected TN-368 cells was accompanied by extensive plasma-membrane blebbing and caspase activation late in infection, both features of apoptosis. Rescue of the p35 locus of AcdefrT continued to result in a reduction in polyhedra and increase in budded virus production in TN-368 cells, but no plasma-membrane blebbing was observed. The mutation was mapped to the FP-25 gene locus. This gene mutation combined with the non-functional p35 was found to be responsible for the cell-blebbing effect observed in AcdefrT-infected TN-368 cells.

The genome sequence of a Polish isolate of Agrotis segetum nucleopolyhedrovirus (AgseNPV-A) was determined and analysed. The circular genome is composed of 147 544 bp and has a G+C content of 45·7 mol%. It contains 153 putative, non-overlapping open reading frames (ORFs) encoding predicted proteins of more than 50 aa, together making up 89·8 % of the genome. The remaining 10·2 % of the DNA constitutes non-coding regions and homologous-repeat regions. One hundred and forty-three AgseNPV-A ORFs are homologues of previously reported baculovirus gene sequences. There are ten unique ORFs and they account for 3 % of the genome in total. All 62 lepidopteran baculovirus genes, including the 29 core baculovirus genes, were found in the AgseNPV-A genome. The gene content and gene order of AgseNPV-A are most similar to those of Spodoptera exigua (Se) multiple NPV and their shared homologous genes are 100 % collinear. Three putative enhancin genes were identified in the AgseNPV-A genome. In phylogenetic analysis, the AgseNPV-A enhancins form a cluster separated from enhancins of the Mamestra species NPVs.

The fowl adenovirus 1 (FAdV-1) isolates PHELPS and OTE are highly similar, but have striking differences in the repeat region of the inverted terminal repeat (ITR). Whilst the repeat region in OTE conforms to the conventional human adenovirus repeat region (5′-CATCATC), that of PHELPS contains guanidine residues at positions 1, 4 and 7 (5′-GATGATG). This implies that the FAdV-1 isolates PHELPS and OTE have either distinct template specificity at replication initiation or, alternatively, a relaxed specificity for replication initiation. In this study, the distinct sequence variation at the origin of DNA replication in the ITRs of the FAdV-1 PHELPS and OTE isolates was confirmed. Sequence analyses of the pTP and Pol genes of both PHELPS and OTE did not reveal differences that could explain the distinct template specificity. Replication assays demonstrated that linear DNA fragments flanked by either 5′-CATCATC or 5′-GATGATG termini replicated in cells upon infection with FAdV-1 OTE and FAdV-1 PHELPS. This was evident from the appearance of DpnI-resistant fragments in a minireplicon assay. From these data, it is concluded that FAdV-1 has relaxed, rather than changed, its template specificity at replication initiation.

Rice tungro bacilliform virus (RTBV) is a double-stranded DNA virus with a single, tissue-specific promoter that is expressed primarily in phloem tissues. Rice transcription factors RF2a and RF2b bind to Box II, a cis element adjacent to the TATA box, and control gene expression from the promoter. Mutations were made in the promoter to delete or mutate Box II and the mutated promoters were fused to a reporter gene; the chimeric genes were expressed in transient BY-2 protoplast assays and in transgenic Arabidopsis plants. The results of these studies showed that Box II is essential to the activity of the RTBV promoter. A chimeric β-glucuronidase (GUS) reporter gene containing the Box II sequence and a minimal promoter derived from the Cauliflower mosaic virus 35S promoter were co-transfected into protoplasts with gene constructs that encoded RF2a or RF2b. The reporter gene produced threefold higher GUS activity when co-transfected with RF2a, and 11-fold higher activity when co-transfected with RF2b, than in the absence of added transcription factors. Moreover, chimeric reporter genes were activated by approximately threefold following induction of expression of the RF2a gene in transgenic Arabidopsis plants. The work presented here and earlier findings show that Box II and its interactions with cognate rice transcription factors, including RF2a and RF2b, are essential to the activity of the RTBV promoter and are probably involved in expression of the RTBV genome during virus replication.