- Volume 83, Issue 1, 2002

The human herpesvirus-8 (HHV-8) locus encoding ORFs 40/41 is a candidate homologue for the Epstein–Barr virus BBLF 2/3 gene, which encodes the putative primase-associated factor. Northern blot data revealed that two transcripts originated from the HHV-8 ORF 40/41 region. The sizes of these transcripts (2.2 and 0.7 kb) suggested that one transcript was the result of a spliced form of ORFs 40 and 41 and the second transcript originated from a region within ORF 41. cDNA sequence and 5′ RACE analysis revealed the removal of an intron between ORFs 40 and 41 and a transcriptional start site 82 nt upstream of ORF 40. The start of transcription for the smaller transcript was mapped to within ORF 41. Regions upstream of the transcriptional start sites were subcloned into a luciferase reporter vector, and transient luciferase assays indicated that distinct promoters drive the expression of each transcript.

The vaccinia virus (VV) F12L gene encodes a 65 kDa protein that is expressed late during infection and is important for plaque formation, EEV production and virulence. Here we have used a recombinant virus (vF12LHA) in which the F12L protein is tagged at the C terminus with an epitope recognized by a monoclonal antibody to determine the location of F12L in infected cells and whether it associates with virions. Using confocal and electron microscopy we show that the F12L protein is located on intracellular enveloped virus (IEV) particles, but is absent from immature virions (IV), intracellular mature virus (IMV) and cell-associated enveloped virus (CEV). In addition, F12L shows co-localization with endosomal compartments and microtubules. F12L did not co-localize with virions attached to actin tails, providing further evidence that actin tails are associated with CEV but not IEV particles. In vΔF12L-infected cells, virus morphogenesis was arrested after the formation of IEV particles, so that the movement of these virions to the cell surface was inhibited and CEV particles were not found. Previously, virus mutants lacking IEV- or EEV-specific proteins were either unable to make IEV particles (vΔF13L and vΔB5R), or were unable to form actin tails after formation of CEV particles (vΔA36R, vΔA33R, vΔA34R). The F12L deletion mutant therefore defines a new stage in the morphogenic pathway and the F12L protein is implicated as necessary for microtubule-mediated egress of IEV particles to the cell surface.

The roles of vaccinia virus (VV) intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV) and their associated proteins in virus spread were investigated. The plaques made by VV mutants lacking individual IEV- or EEV-specific proteins (vΔA33R, vΔA34R, vΔA36R, vΔA56R, vΔB5R, vΔF12L and vΔF13L) were compared in the presence of IMV- or EEV-neutralizing antibodies (Ab). Data presented show that for long-range spread, the comet-shaped plaques of VV were caused by the unidirectional spread of EEV probably by convection currents, and for cell-to-cell spread, VV uses a combination of Ab-resistant and Ab-sensitive pathways. Actin tails play a major role in the Ab-resistant pathway, but mutants such as vΔA34R and vΔA36R that do not make actin tails still spread from cell to cell in the presence of Ab. Most strikingly, the Ab-resistant pathway was abolished when the A33R gene was deleted. This effect was not due to alterations in the efficiency of neutralization of EEV made by this mutant, nor due to a deficiency in IMV wrapping to form IEV, which was indispensable for EEV formation by vΔA33R and vΔA34R. We suggest a role for A33R in promoting Ab-resistant cell-to-cell spread of virus. The roles of the different virus forms in the VV life-cycle are discussed.

The SPAn232 virus (SPAnv) was isolated from sentinel mice in the forest of Cotia, São Paulo, Brazil. It was grouped originally as a Cotia virus (CV) sample due to serological cross-reaction with the latter. However, SPAnv presented genetic characteristics that differed from CV and indicated that SPAnv is a member of the vaccinia virus (VV) subgroup. SPAnv showed a HindIII-digested DNA pattern similar to those of the WR and Lister strains of VV. Also, SPAnv presented genes homologous to the vaccinia growth factor, thymidine kinase and A-type inclusion (ATI) genes from VV. RFLP analysis of the SPAnv ATI homologous gene indicated that the virus belongs to the VV group. Nucleotide sequences from SPAnv genes showed up to 99% similarity with the same genes from VV. Such a relationship was confirmed visually through the drawing of phylogenetic trees. The results point out the occurrence of a VV strain that is possibly in active circulation in the forests of Southeast Brazil.

White spot syndrome virus (WSSV) infects penaeid shrimp and other crustaceans. The WSSV virion consists of an enveloped rod-shaped nucleocapsid enclosing a large circular double-stranded DNA genome of 293 kbp. The virion envelope contains two major proteins of 28 (VP28) and 19 kDa (VP19) and the nucleocapsid consists of three major proteins of 26 (VP26), 24 (VP24) and 15 kDa (VP15). Study on the morphogenesis of the WSSV particle requires the genomic identification and chemical characterization of these WSSV virion proteins. An internal amino acid sequence of envelope protein VP19 was obtained by amino acid sequencing and used to locate the VP19 open reading frame of this protein on the genome, as WSSV ORF182. VP19 contained two putative transmembrane domains, which may anchor this protein in the WSSV envelope. Similarly, the gene for VP15 was located on the WSSV genome as ORF109. N-terminal amino acid sequencing on VP15 suggested that this protein was expressed from the second ATG of its ORF and the first methionine is lost by N-terminal protein processing. The 15 kDa protein is very basic and is a candidate DNA-binding protein in the WSSV nucleocapsid. None of the five major structural WSSV proteins appear to be glycosylated, which is an unusual feature among enveloped animal viruses.

Potato virus X (PVX) and narcissus mosaic virus (NMV) were studied using vibrational Raman optical activity (ROA) in order to obtain new information on the structures of their coat protein subunits. The ROA spectra of the two intact virions are very similar to each other and similar to that of tobacco mosaic virus (TMV) studied previously, being dominated by signals characteristic of proteins with helix bundle folds. In particular, PVX and NMV show strong positive ROA bands at ∼1340 cm−1 assigned to hydrated α-helix and perhaps originating in surface exposed helical residues, together with less strong positive ROA intensity in the range ∼1297–1312 cm−1 assigned to α-helix in a more hydrophobic environment and perhaps originating in residues at helix–helix interfaces. The positive ∼1340 cm−1 ROA band of TMV is less intense than those of PVX and NMV, suggesting that TMV contains less hydrated α-helix. Small differences in other spectral regions reflect differences in some loop, turn and side-chain compositions and conformations among the three viruses. A pattern recognition program based on principal component analysis of ROA spectra indicates that the coat protein subunit folds of PVX and NMV may be very similar to each other and similar to that of TMV. These results suggest that PVX and NMV may have coat protein subunit structures based on folds similar to the TMV helix bundle and hence that the helical architecture of the PVX and NMV particles may be similar to that of TMV but with different structural parameters.