Structure and Expression of Large (+)RNA Genomes of Viruses of Higher Eukaryotes.

Viral positive-sense RNA genomes evolve rapidly due to the high mutation rates during replication and RNA recombination, which allowing the viruses to acquire and modify genes for their adaptation. The size of RNA genome is limited by several factors, including low fidelity of RNA polymerases and packaging constraints. However, the 12-kb size limit is exceeded in the two groups of eukaryotic (+)RNA viruses - animal nidoviruses and plant closteroviruses. These virus groups have several traits in common. Their genomes contain 5'-proximal genes that are expressed via ribosomal frameshifting and encode one or two papain-like protease domains, membrane-binding domain(s), methyltransferase, RNA helicase, and RNA polymerase. In addition, some nidoviruses (i.e., coronaviruses) contain replication-associated domains, such as proofreading exonuclease, putative primase, nucleotidyltransferase, and endonuclease. In both nidoviruses and closteroviruses, the 3'-terminal part of the genome contains genes for structural and accessory proteins expressed via a nested set of coterminal subgenomic RNAs. Coronaviruses and closteroviruses have evolved to form flexuous helically symmetrical nucleocapsids as a mean to resolve packaging constraints. Since phylogenetic reconstructions of the RNA polymerase domains indicate only a marginal relationship between the nidoviruses and closteroviruses, their similar properties likely have evolved convergently, along with the increase in the genome size.

Molecular Alliance of Lymantria dispar Multiple Nucleopolyhedrovirus and a Short Unmodified Antisense Oligonucleotide of Its Anti-Apoptotic IAP-3 Gene: A Novel Approach for Gypsy Moth Control.

Baculovirus IAP (inhibitor-of-apoptosis) genes originated by capture of host genes. Unmodified short antisense DNA oligonucleotides (oligoDNAs) from baculovirus IAP genes can down-regulate specific gene expression profiles in both baculovirus-free and baculovirus-infected insects. In this study, gypsy moth (Lymantria dispar) larvae infected with multiple nucleopolyhedrovirus (LdMNPV), and LdMNPV-free larvae, were treated with oligoDNA antisense to the RING (really interesting new gene) domain of the LdMNPV IAP-3 gene. The results with respect to insect mortality, biomass accumulation, histological studies, RT-PCR, and analysis of DNA apoptotic fragmentation suggest that oligoRING induced increased apoptotic processes in both LdMNPV-free and LdMNPV-infected insect cells, but were more pronounced in the latter. These data open up possibilities for promising new routes of insect pest control using antisense phosphodiester DNA oligonucleotides.

Completion of the mapping of transcription start sites for the five-gene block subgenomic RNAs of Beet yellows Closterovirus and identification of putative subgenomic promoters.

In the positive-sense RNA genome of Beet yellows Closterovirus (BYV), the 3'-terminal open reading frames (ORFs) 2-8 are expressed as a nested set of subgenomic (sg) RNAs. ORFs 2-6, coding for the structural and movement proteins, form a 'five-gene block' conserved in closteroviruses. We mapped the 5'-end of the ORF 4 sgRNA, which encodes the p64 protein, at adenosine-11169 in the BYV genome. This completes the mapping of the transcription start sites for the five-gene block sgRNAs of BYV. Computer-assisted analysis of the sequences upstream of BYV ORFs 2, 3, 4, 5, and 6 revealed two conserved motifs, which might constitute the subgenomic promoter elements. These motifs are conserved in the equivalent positions upstream of three orthologous genes of Citrus tristeza Closterovirus and two orthologous genes of Beet yellow stunt Closterovirus.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell.

Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses.

In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-µm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of "virus factories" in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).

Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus.

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus.

Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and helicase (HEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragonia expansa plants. MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous structures, the 'BYV-type vesicles', thus suggesting that the closterovirus MT-like and HEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the recombinant MT and HEL fragments that had been used as immunogens showed that 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686-692 in the BYV 1a protein), and 1C4 reacted with the DDPF epitope (aa 2493-2496). These epitopes apparently reside on the exposed parts of the membrane-associated molecules of the closterovirus MT-like and HEL-like proteins. Two other epitopes determined for the MT MAbs that were nonreactive in the immunogold labelling, namely TMVTPGEL (aa 750-757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806-813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.