26 research outputs found
Investigation of Interactions of the Rubella Virus P150 Replicase Protein with Host Cell Proteins in Infected Cells
Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis
Lack of Processing of the Expressed ORF1 Gene Product of Hepatitis E Virus
Background: Proteolytic processing is a common mechanism among plus strand RNA viruses and the replicases of all plus strand RNA viruses of animals thus far characterized undergo such processing. The replicase proteins of hepatitis E virus (HEV) are encoded by ORF1. A previous report published by our group [1] provided data that processing potentially occurred when ORF1 (Burma strain; genotype 1) was expressed using a vaccinia virus-based expression system.
Findings: To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed. When ORF1 from an HEV infectious cDNA clone (US swine strain; genotype 3) was expressed using the plasmid-based system, the only species detected was the 185 kDa precursor of ORF1. A putative papain-like cysteine protease [2] had been predicted within ORF1 using the original HEV genomic sequence. However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved.
Conclusions: The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard
Sexual Transmission of XMRV: A Potential Infection Route
Although XMRV dissemination in humans is a matter of debate, the prostate of select patients seem to harbor XMRV, which raises questions about its potential route of transmission. We established a model of infection in rhesus macaques inoculated with XMRV. In spite of the intravenous inoculation, all infected macaques exhibited readily detectable XMRV signal in the reproductive tract of all 4 males and 1 female during both acute and chronic infection stages. XMRV showed explosive growth in the acini of prostate during acute but not chronic infection. In seminal vesicles, epididymis, and testes, XMRV protein production was detected throughout infection in interstitial or epithelial cells. In the female monkey, epithelial cells in the cervix and vagina were also positive for XMRV gag. The ready detection of XMRV in the reproductive tract of male and female macaques infected intravenously suggests the potential for sexual transmission for XMRV
Antibody Responses against Xenotropic Murine Leukemia Virus-Related Virus Envelope in a Murine Model
Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV.Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations.Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans
Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1) virus have higher neuraminidase activities than the spherical wild-type.
Influenza virus exhibits two morphologies - spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo
Drug Sensitivity of Vaccine-Derived Rubella Viruses and Quasispecies Evolution in Granulomatous Lesions of Two Ataxia-Telangiectasia Patients Treated with Nitazoxanide.
A strong association between rubella virus (RuV) and chronic granulomas, in individuals with inborn errors of immunity, has been recently established. Both the RA27/3 vaccine and wild-type RuV strains were highly sensitive to a broad-spectrum antiviral drug, nitazoxanide (NTZ), in vitro. However, NTZ treatment, used as a salvage therapy, resulted in little or no improvements of RuV-associated cutaneous granulomas in patients. Here, we report investigations of possible causes of treatment failures in two ataxia-telangiectasia patients. Although a reduction in RuV RNA in skin lesions was detected by real-time RT-PCR, live immunodeficiency-related vaccine-derived rubella viruses (iVDRV) were recovered from granulomas, before and after the treatments. Tizoxanide, an active NTZ metabolite, inhibited replications of all iVDRVs in cultured A549 cells, but the 50% and 90% inhibitory concentrations were 10-40 times higher than those for the RA27/3 strain. There were no substantial differences in iVDRV sensitivities, neither before nor after treatments. Analysis of quasispecies in the E1 gene, a suspected NTZ target, showed no effect of NTZ treatments on quasispecies' complexity in lesions. Thus, failures of NTZ therapies were likely due to low sensitivities of iVDRVs to the drug, and not related to the emergence of resistance, following long-term NTZ treatments
128 HAU of rPR8wt, rPR8 M1 N87S and rPR8 M1 R101G viruses comprise comparable infectious titers and genome copies.
a<p>rPR8wt to rPR8 M1 N87S comparison (p = 0.38), rPR8wt to rPR8 M1 R101G comparison (p = 0.40). A two-tailed Student <i>t</i>-test was used to assess significance.</p>b<p>rPR8wt to rPR8 M1 N87S comparison (p = 0.23), rPR8wt to rPR8 M1 R101G comparison (p = 0.10). To assess significance, a two-tailed Student <i>t</i>-test was applied to values of 2<sup>(−Cq)</sup>.</p><p>128 HAU of rPR8wt, rPR8 M1 N87S and rPR8 M1 R101G viruses comprise comparable infectious titers and genome copies.</p
No difference in virion stability at an elevated temperature was observed between rPR8wt, rPR8 M1 N87S, and rPR8 M1 R101G viruses.
<p>Each virus was diluted to 1×10<sup>6</sup> PFU and incubated in triplicate at 50°C for one of the following lengths of time: 0, 15, 30, 60, and 120 min. Results shown are the average of four separate assays performed in triplicate (thus n = 12). The titers of each mutant virus were compared to that of the wt virus at each time point using Student’s <i>t</i> test. All p values were >0.05 except that obtained for wt vs. N87S viruses at the 0 h time point (p = 0.0094). The dotted line indicates the limit of detection.</p
rPR8 M1 R101G and rPR8 M1 N87S viruses have higher neuraminidase activity than rPR8wt virus.
<p>A) Neuraminidase enzyme kinetics. Virus input was standardized to 5×10<sup>5</sup> PFU and concentrations of MUNANA substrate ranging from 1.17 µM to 150 µM were used. Fluorescence generated at each time point (every minute over the course of 1 hour) was detected using a Biotek Synergy H1 plate reader. The resulting fluorescence curves were then fitted to the Michaelis-Menton equation. B) That equivalent amounts of each virus were used in the MUNANA assay was confirmed by RT-qPCR for the viral NP segment. The arithmetic mean (n = 3) and standard deviation of 2<sup>(−Cq)</sup> values were calculated and then converted back to a C<sub>q</sub> scale by taking the log<sub>2</sub>. Two biological replicates of each virus are included in white and grey bars; each biological replicate comprised three technical replicates.</p