Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were inserted between the repeats. The inner repeat and exogenous insert were positioned in frame with the native protein-encoding sequences but were separated from them by an in-frame "2A-like" sequence element that specifies a cotranslational "stop/continue" event releasing the exogenous polypeptide from the essential MRV protein. This design preserves a terminal region of the MRV genome segment with essential activities in RNA packaging, assortment, replication, transcription, and/or translation and alters the encoded MRV protein to a limited degree. Recovery of rMRVs with longer inserts was made more efficient by wobble-mutagenizing both the inner repeat and the exogenous insert, which possibly helped via respective reductions in homologous recombination and RNA structure. Immunogenicity of a 300-aa portion of the simian immunodeficiency virus Gag protein expressed in mice by an L1-modified rMRV was confirmed by detection of Gag-specific T-cell responses. The engineering strategy was further used for mapping the minimal 5'-terminal region essential to MRV genome segment S3.

Effects of viscogens on RNA transcription inside reovirus particles.

The dsRNA genome of mammalian reovirus (MRV), like the dsDNA genomes of herpesviruses and many bacteriophages, is packed inside its icosahedral capsid in liquid-crystalline form, with concentrations near or more than 400 mg/ml. Viscosity in such environments must be high, but the relevance of viscosity for the macromolecular processes occurring there remains poorly characterized. Here, we describe the use of simple viscogens, glycerol and sucrose, to examine their effects on RNA transcription inside MRV core particles. Transcription inside MRV cores was strongly inhibited by these agents and to a greater extent than either predicted by theory or exhibited by a nonencapsidated transcriptase, suggesting that RNA transcription inside MRV cores is unusually sensitive to viscogen effects. The elongation phase of transcription was found to be a primary target of this inhibition. Similar results were obtained with particles of a second dsRNA virus, rhesus rotavirus, from a divergent taxonomic subfamily. Polymeric viscogens such as polyethylene glycol also inhibited RNA transcription inside MRV cores, but in a size-limited manner, suggesting that diffusion through channels in the MRV core is required for their activity. Modeling of the data suggested that the inherent intracapsid viscosity of both reo- and rotavirus is indeed high, two to three times the viscosity of water. The capacity for quantitative comparisons of intracapsid viscosity and effects of viscogens on macromolecular processes in confined spaces should be similarly informative in other systems.

Recruitment of cellular clathrin to viral factories and disruption of clathrin-dependent trafficking.

The viral factories of mammalian reovirus (MRV) are cytoplasmic structures that serve as sites of viral genome replication and particle assembly. A 721-aa MRV non-structural protein, µNS, forms the factory matrix and recruits other viral proteins to these structures. In this report, we show that µNS contains a conserved C-proximal sequence (711-LIDFS-715) that is similar to known clathrin-box motifs and is required for recruitment of clathrin to viral factories. Clathrin recruitment by µNS occurs independently of infecting MRV particles or other MRV proteins. Ala substitution for a single Leu residue (mutation L711A) within the putative clathrin-binding motif of µNS inhibits clathrin recruitment, but does not prevent formation or expansion of viral factories. Notably, clathrin-dependent cellular functions, including both endocytosis and secretion, are disrupted in cells infected with MRV expressing wild-type, but not L711A, µNS. These results identify µNS as a novel adaptor-like protein that recruits cellular clathrin to viral factories, disrupting normal functions of clathrin in cellular membrane trafficking. To our knowledge, this is the only viral or bacterial protein yet shown to interfere with clathrin functions in this manner. The results additionally establish a new approach for studies of clathrin functions, based on µNS-mediated sequestration.

Clinical isolates of Trichomonas vaginalis concurrently infected by strains of up to four Trichomonasvirus species (Family Totiviridae).

Trichomonas vaginalis, which causes the most common nonviral sexually transmitted disease worldwide, is itself commonly infected by nonsegmented double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae. To date, cDNA sequences of one or more strains of each of three trichomonasvirus species have been reported, and gel electrophoresis showing several different dsRNA molecules obtained from a few T. vaginalis isolates has suggested that more than one virus strain might concurrently infect the same parasite cell. Here, we report the complete cDNA sequences of 3 trichomonasvirus strains, one from each of the 3 known species, infecting a single, agar-cloned clinical isolate of T. vaginalis, confirming the natural capacity for concurrent (in this case, triple) infections in this system. We furthermore report the complete cDNA sequences of 11 additional trichomonasvirus strains, from 4 other clinical isolates of T. vaginalis. These additional strains represent the three known trichomonasvirus species, as well as a newly identified fourth species. Moreover, 2 of these other T. vaginalis isolates are concurrently infected by strains of all 4 trichomonasvirus species (i.e., quadruple infections). In sum, the full-length cDNA sequences of these 14 new trichomonasviruses greatly expand the existing data set for members of this genus and substantiate our understanding of their genome organizations, protein-coding and replication signals, diversity, and phylogenetics. The complexity of this virus-host system is greater than has been previously well recognized and suggests a number of important questions relating to the pathogenesis and disease outcomes of T. vaginalis infections of the human genital mucosa.