Comparative mutational analysis of cis-acting RNA signals for translational frameshifting in HIV-1 and HTLV-2

Human immunodeficiency virus type 1 (HIV-1) and human T cell leukemia virus type II (HTLV-2) use a similar mechanism for –1 translational frameshifting to overcome the termination codon in viral RNA at the end of the gag gene. Previous studies have identified two important RNA signals for frameshifting, the slippery sequence and a downstream stem–loop structure. However, there have been somewhat conflicting reports concerning the individual contributions of these sequences. In this study we have performed a comprehensive mutational analysis of the cis-acting RNA sequences involved in HIV-1 gag–pol and HTLV-2 gag–pro frameshifting. Using an in vitro translation system we determined frameshifting efficiencies for shuffled HIV-1/HTLV-2 RNA elements in a background of HIV-1 or HTLV-2 sequences. We show that the ability of the slippery sequence and stem–loop to promote ribosomal frameshifting is influenced by the flanking upstream sequence and the nucleotides in the spacer element. A wide range of frameshift efficiency rates was observed for both viruses when shuffling single sequence elements. The results for HIV-1/HTLV-2 chimeric constructs represent strong evidence supporting the notion that the viral wild-type sequences are not designed for maximal frameshifting activity but are optimized to a level suited to efficient viral replication.

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

Functional implications of protein-protein interactions in icosahedral viruses.

Biological processes often require that a single gene product participate in multiple types of molecular interactions. Viruses with quasiequivalent capsids provide an excellent paradigm for studying such phenomena because identical protein subunits are found in different structural environments. Differences in subunit joints may be controlled by protein segments, duplex or single-stranded RNA, metal ions, or some combination of these. Each of the virus groups examined display a distinctive mechanism for switching interface interactions, illustrating the magnitude of options that are likely to be found in other biological systems. In addition to determining capsid morphology, assembly controls the timing of autocatalytic maturation cleavage of the viral subunits that is required for infectivity in picorna-, noda-, and tetraviruses. The mechanism of assembly-dependent cleavage is conserved in noda- and tetraviruses, although the quaternary structures of the capsids are different as are the molecular switches that control subunit interfaces. The function of the cleavage in picorna-, noda-, and tetraviruses is probably to release polypeptides that participate in membrane translocation of RNA.

Broad resistance to plant viruses in transgenic plants conferred by antisense inhibition of a host gene essential in S-adenosylmethionine-dependent transmethylation reactions.

S-Adenosylhomocysteine hydrolase (SAHH) is a key enzyme in transmethylation reactions that use S-adenosylmethionine as the methyl donor. Because of the importance of SAHH in a number of S-adenosylmethionine-dependent transmethylation reactions, particularly the 5' capping of mRNA during viral replication, SAHH has been considered as a target of potential antiviral agents against animal viruses. To test the possibility of engineering a broad type of resistance to plant viruses, we expressed the antisense RNA for tobacco SAHH in transgenic tobacco plants. As expected, transgenic plants constitutively expressing an anti-sense SAHH gene showed resistance to infection by various plant viruses. Among those plants, about half exhibited some level of morphological change (typically stunting). Analysis of the physiological change in those plants showed that they contained excess levels of cytokinin. Because cytokinin has been found to induce acquired resistance, there is also a strong possibility that the observed resistance was induced by cytokinin.

Mouse mammary tumor viruses with functional superantigen genes are selected during in vivo infection.

Mouse mammary tumor virus (MMTV) encodes a superantigen that is important for viral infectivity in vivo. To determine whether superantigen function was required for infection by milk-borne MMTV, we created HYB PRO/Cla transgenic mice. These mice produced a full-length, packaged viral RNA with a frameshift mutation that caused premature termination of the superantigen protein. Young HYB PRO/Cla mice showed no deletion of their cognate V beta 14+ T cells, although they shed virus in their milk. The nontransgenic offspring of the HYB PRO/Cla mice were infected with this virus, since transgene-specific viral transcripts were detected in their mammary glands. Surprisingly, these offspring demonstrated the progressive deletion of V beta 14+ T cells characteristic of exogenous MMTV (C3H) infection. Sequence analysis demonstrated that these newly acquired viruses had reconstituted superantigen open reading frames resulting from recombination between the HYB PRO/Cla and endogenous Mtv-1 proviral RNAs. Thus, there is selection during the infection process for MMTVs with functional superantigen genes.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Highly accurate RNA and DNA sequencing: Application to longstanding questions in aging and cancer

Thesis (Ph.D.)--University of Washington, 2016-06

Presence of a novel small RNA-containing virus in a laboratory culture of the endoparasitic wasp Venturia canescens (Hymenoptera : Ichneumonidae)

Parasitoid wasps use a variety of mechanisms to alter their host's physiology to the benefit of the developing endoparasite inside the host larva. Association of certain wasps with viruses and virus-like particles (VLPs) that contribute to their success in parasitism is one of the fascinating evolutionary adaptations conferring active or passive protection for the endoparasite from the host immune system. Venturia canescens has been shown to produce VLPs that provide protection for the developing parasitoid egg inside the host, Ephestia kuehniella. Here, we report on the presence of a novel small RNA-containing virus from V. canescens, designated as VcSRV, occurring in the ovaries of the wasp. The virus particles are found together with VcVLPs in the lumen of the calyx region of the ovaries and are injected together with the egg and VcVLPs into E kuehniella larvae where they enter hemocytes. Alignment of the RNA-dependent RNA polymerase gene of VcSRV indicates that the virus most likely belongs to the recently described genus Iflavirus. (c) 2004 Elsevier Ltd. All rights reserved.

A metagenomic comparison of endemic viruses from broiler chickens with runting stunting syndrome and from normal birds

Runting-stunting syndrome (RSS) in broiler chickens is an enteric disease that causes significant economic losses to poultry producers worldwide due to elevated feed conversion ratios, decreased body weight during growth, and excessive culling. Of specific interest are the viral agents associated with RSS which have been difficult to fully characterise to date. Past research into the aetiology of RSS has implicated a wide variety of RNA and DNA viruses however, to date, no individual virus has been identified as the main agent of RSS and the current opinion is that it may be caused by a community of viruses, collectively known as the virome. This paper attempts to characterise the viral pathogens associated with 2 – 3 week old RSS-affected and unaffected broiler chickens using next-generation sequencing and comparative metagenomics. Analysis of the viromes identified a total of 20 DNA & RNA viral families, along with 2 unidentified categories, comprised of 31 distinct viral genera and 7 unclassified genera. The most abundant viral families identified in this study were the Astroviridae, Caliciviridae, Picornaviridae, Parvoviridae, Coronaviridae, Siphoviridae, and Myoviridae. This study has identified historically significant viruses associated with the disease such as chicken astrovirus, avian nephritis virus, chicken parvovirus, and chicken calicivirus along with relatively novel viruses such as chicken megrivirus and sicinivirus 1 and will help expand the knowledge related to enteric disease in broiler chickens, provide insights into the viral constituents of a healthy avian gut, and identify a variety of enteric viruses and viral communities appropriate for further study.

Investigation of the functional roles of host cell proteins involved in Coronavirus infection using highly specific and scalable RNA interference (RNAi) approach

Since its identification in the 1990s, the RNA interference (RNAi) pathway has proven extremely useful in elucidating the function of proteins in the context of cells and even whole organisms. In particular, this sequence-specific and powerful loss-of-function approach has greatly simplified the study of the role of host cell factors implicated in the life cycle of viruses. Here, we detail the RNAi method we have developed and used to specifically knock down the expression of ezrin, an actin binding protein that was identified by yeast two-hybrid screening to interact with the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) spike (S) protein. This method was used to study the role of ezrin, specifically during the entry stage of SARS-CoV infection.

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims: Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies. Method: A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples. Conclusions: The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not. Significance and Impact of the Study: The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.