- Volume 85, Issue 1, 2004

We have examined the subcellular localization properties of human adenovirus 2 (HAdV-2) preMu and mature Mu (pX) proteins as fusions with enhanced green fluorescence protein (EGFP). We determined that preMu is exclusively a nucleolar protein with a single nucleolar accumulation signal within the Mu sequence. In addition, we noted that both preMu–EGFP and Mu–EGFP are excluded from adenovirus DNA-binding protein (DBP)-rich replication centres in adenovirus-infected cells. Surprisingly, we observed that cells in which preMu–EGFP (but not Mu–EGFP) is transiently expressed prior to or shortly after infection with Ad2 did not express late adenovirus genes. Further investigation suggested this might be due to a failure to express pre-terminal protein (preTP) from the E2 region, despite expression of another E2 protein, DBP. Deletion mutagenesis identified a highly conserved region in the C terminus of preMu responsible for these observations. Thus our data suggest that preMu may play a role in modulating accumulation of proteins from the E2 region.

Knowledge of virus genes under positive selection pressure can help identify molecular determinants of species-specific virulence or host range without prior knowledge of the mechanisms governing host range and virulence. Towards this end, codon-based models of substitution were used in a maximum-likelihood approach to analyse selection pressures acting on 83 genes of group I nucleopolyhedroviruses (NPVs). Evidence for positive selection was found for nine genes: ac38, ac66, arif-1, lef-7, lef-10, lef-12, odv-e18, odv-e56 and vp80. The baculovirus DNA helicase gene (dnahel) was not found to be positively selected using models that allowed the intensity of selection pressure to vary among codon sites. Further analysis with a method that allows selection pressure intensity to vary among lineages suggests that positive selection may have occurred in dnahel during the divergence of Bombyx mori NPV and the NPVs of Autographa californica and Rachiplusia ou. NPV genes that have undergone positive selection may modulate the ability of different NPVs to replicate efficiently in cells (lef-7, lef-10, lef-12) or to establish primary infection of the midgut (odv-e18, odv-e56) of different host species.

Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) is one of the most successful biological insecticides. In this study, we cloned and sequenced a 12·5 kbp BamHI-D restriction endonuclease fragment of the AgMNPV isolate 2D genome that includes the gp64 gene. We compared this highly conserved region with that of other baculoviruses. AgMNPV contained two genes, p22.2 and v-trex, in common with Choristoneura fumiferana MNPV (CfMNPV) that were not present in other baculoviruses. The v-trex gene has homology to a eukaryotic 3′ repair exonuclease and appears to have been acquired from an invertebrate host. The v-trex gene product has the potential to be involved in virus recombination or UV-light tolerance. Multigene phylogenetic analysis suggested that AgMNPV is most closely related to Orgyia pseudotsugata MNPV (OpMNPV). AgMNPV differed from other group I NPVs in that ChiA and v-cath gene homologues were missing from the region downstream of the gp64 gene. Proteinase assays and genetic probes suggest the v-cath gene is absent from AgMNPV.

The coat protein (CP) of Alfalfa mosaic virus (AMV) is required to initiate infection by the viral tripartite RNA genome whereas infection by the tripartite Brome mosaic virus (BMV) genome is independent of CP. AMV CP stimulates translation of AMV RNA in vivo 50- to 100-fold. The 3′ untranslated region (UTR) of the AMV subgenomic CP messenger RNA 4 contains at least two CP binding sites. A CP binding site in the 3′-terminal 112 nucleotides of RNA 4 was found to be required for efficient translation of the RNA whereas an upstream binding site was not. Binding of CP to the AMV 3′ UTR induces a conformational change of the RNA but this change alone was not sufficient to stimulate translation. CP mutant R17A is unable to bind to the 3′ UTR and translation in vivo of RNA 4 encoding this mutant occurs at undetectable levels. Replacement of the 3′ UTR of this mutant RNA 4 by the 3′ UTR of BMV RNA 4 restored translation of R17A-CP to wild-type levels. Apparently, the BMV 3′ UTR stimulates translation independently of CP. AMV CP mutant N199 is defective in the formation of CP dimers and did not stimulate translation of RNA 4 in vivo although the mutant CP did bind to the 3′ UTR. The finding that N199-CP does not promote AMV infection corroborates the notion that the requirement of CP in the inoculum reflects its role in translation of the viral RNAs.

Tobacco etch potyvirus (TEV) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (HC-Pro). To produce a biologically active form of recombinant TEV HC-Pro protein, heterologous expression in the methylotrophic yeast Pichia pastoris was used. A cDNA encoding the TEV HC-Pro region, fused to a Saccharomyces cerevisiae α-mating factor secretory peptide coding region, was inserted into the P. pastoris genome using a modified version of the pPIC9 vector. The expressed TEV HC-Pro protein was obtained directly from culture medium of recombinant yeast colonies; it was able to interact with TEV particles in a protein overlay binding assay, and also to assist aphid transmission of purified TEV particles to plants using the aphid Myzus persicae as vector. Our results indicate that P. pastoris provides a rapid and low-cost heterologous expression system that can be used to obtain biologically active potyvirus HC-Pro protein for in vitro transmission assays.

The protein encoded by the first gene of the triple gene block (TGBp1) of potexviruses is required for movement of the viruses. It has been reported that single Arg→Ala substitutions at position 11, 16 or 21 of TGBp1 of Bamboo mosaic virus (BaMV) eliminate its RNA-binding activity, while substitutions at position 16 or 21 only affect its NTPase activity ( Liou et al., Virology 277, 336–344, 2000 ). However, it remains unclear whether these Arg→Ala substitutions also affect the movement of BaMV in plants. To address this question, six mutants of BaMV, each containing either a single- or a double-alanine substitution at Arg-11, Arg-16 and Arg-21 of TGBp1, were constructed and used to infect Chenopodium quinoa and Nicotiana benthamiana. We found that all of the BaMV mutants were able to replicate in protoplasts of N. benthamiana. However, only the mutant with an Arg-11→Ala substitution in TGBp1 remained capable of movement from cell to cell in plants. Mutants with Arg-16, Arg-21 or both Arg-16 and Arg-21 of TGBp1 replaced with alanine were defective in virus movement. This defect was suppressed when a wild-type TGBp1 allele was co-introduced into the cells using a novel satellite replicon. The ability to trans-complement the movement defect by the wild-type TGBp1 strongly suggests that the Arg→Ala substitution at position 16 or 21 of TGBp1, which diminishes the RNA-binding and NTPase activities of TGBp1, also eliminates the capability of BaMV to move from cell to cell in host plants.

We report that the replication-deficient DY strain of transmissible mink encephalopathy (TME) can delay disease caused by the pathogenic HY TME strain. In this study, competition between the HY and DY TME agents was investigated following superinfection of the sciatic nerve and peritoneal cavity. Initially, DY TME infection was examined in the absence of superinfection and it was found that inoculation into the brain and sciatic nerve resulted in prion disease and PrPSc deposition in brain but not lymphoreticular tissues. Conversely, intraperitoneal inoculation of the DY TME agent did not result in clinical symptoms, DY TME agent replication or PrPSc deposition 400–600 days after infection. These findings indicate that the DY TME agent does not replicate in secondary lymphoid organs and is non-pathogenic when neuroinvasion is dependent on prior infection of the lymphoreticular system. However, intraperitoneal inoculation of the DY TME agent at 60 days, but not at 30 days, prior to intraperitoneal inoculation of the HY TME agent resulted in an extension of the HY TME incubation period. Inoculation of the DY TME agent into the sciatic nerve at 60 days prior to intrasciatic nerve inoculation of the HY TME agent did not delay the incubation period of HY TME. The ability of the DY TME agent to delay HY TME infection following extraneural inoculation, but not neural infection, suggests that HY and DY TME agent competition can occur in a common replication site whose cellular location precedes infection of both the lymphoreticular and peripheral nervous systems.