Complete genome organization of American hop latent virus and its relationship to carlaviruses

The complete genomic sequence of American hop latent virus (AHLV; genus Carlavirus) was determined. The genome consists of 8,601 nucleotides plus a 3′-polyadenylate tail. The genome encompasses six potential open reading frames (ORF) in the positive sense, and their organization is typical of other carlaviruses. Analysis of the coat protein coding sequence at both the nucleic acid level and the amino acid level indicates that AHLV is only remotely related to the other carlaviruses known to infect common hop. Polyclonal antibodies were produced against the bacterially expressed coat protein of AHLV. These antibodies differentiated between AHLV and other carlaviruses of hop

Movement Protein Pns6 of Rice dwarf phytoreovirus Has Both ATPase and RNA Binding Activities

Cell-to-cell movement is essential for plant viruses to systemically infect host plants. Plant viruses encode movement proteins (MP) to facilitate such movement. Unlike the well-characterized MPs of DNA viruses and single-stranded RNA (ssRNA) viruses, knowledge of the functional mechanisms of MPs encoded by double-stranded RNA (dsRNA) viruses is very limited. In particular, many studied MPs of DNA and ssRNA viruses bind non-specifically ssRNAs, leading to models in which ribonucleoprotein complexes (RNPs) move from cell to cell. Thus, it will be of special interest to determine whether MPs of dsRNA viruses interact with genomic dsRNAs or their derivative sRNAs. To this end, we studied the biochemical functions of MP Pns6 of Rice dwarf phytoreovirus (RDV), a member of Phytoreovirus that contains a 12-segmented dsRNA genome. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5′- and 3′- terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the RNA binding and ATPase activity sites of Pns6 at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions

Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels

Structural Analysis of the −1 Ribosomal Frameshift Elements in Giardiavirus mRNA

The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a −1 ribosomal frameshift in a region where gag and pol open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5′ 631-nt cDNA and the out-of-frame luciferase gene that required a −1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or −1 frame, located directly before or after the heptamer, implicated the latter as the site for the −1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3′ end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a −1 ribosomal frameshift in the GLV transcript