Mutational analysis of residues involved in nucleotide and divalent cation stabilization in the rotavirus RNA-dependent RNA polymerase catalytic pocket

AbstractThe rotavirus RNA-dependent RNA polymerase (RdRp), VP1, contains canonical RdRp motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential import to nucleotide and divalent cation stabilization, we aligned VP1 of divergent rotaviruses and the structural homolog reovirus λ3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRp motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRp activity to particle assembly

Hepatitis C virus RNA replication depends on specific cis- and trans-acting activities of viral nonstructural proteins

Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3-5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3-5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally

Rheumatoid arthritis - clinical aspects: 134. Predictors of Joint Damage in South Africans with Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA) causes progressive joint damage and functional disability. Studies on factors affecting joint damage as clinical outcome are lacking in Africa. The aim of the present study was to identify predictors of joint damage in adult South Africans with established RA. Methods: A cross-sectional study of 100 black patients with RA of >5 years were assessed for joint damage using a validated clinical method, the RA articular damage (RAAD) score. Potential predictors of joint damage that were documented included socio-demographics, smoking, body mass index (BMI), disease duration, delay in disease modifying antirheumatic drug (DMARD) initiation, global disease activity as measured by the disease activity score (DAS28), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and autoantibody status. The predictive value of variables was assessed by univariate and stepwise multivariate regression analyses. A p value <0.05 was considered significant. Results: The mean (SD) age was 56 (9.8) years, disease duration 17.5 (8.5) years, educational level 7.5 (3.5) years and DMARD lag was 9 (8.8) years. Female to male ratio was 10:1. The mean (SD) DAS28 was 4.9 (1.5) and total RAAD score was 28.3 (12.8). The mean (SD) BMI was 27.2 kg/m2 (6.2) and 93% of patients were rheumatoid factor (RF) positive. More than 90% of patients received between 2 to 3 DMARDs. Significant univariate predictors of a poor RAAD score were increasing age (p = 0.001), lower education level (p = 0.019), longer disease duration (p < 0.001), longer DMARD lag (p = 0.014), lower BMI (p = 0.025), high RF titre (p < 0.001) and high ESR (p = 0.008). The multivariate regression analysis showed that the only independent significant predictors of a higher mean RAAD score were older age at disease onset (p = 0.04), disease duration (p < 0.001) and RF titre (p < 0.001). There was also a negative association between BMI and the mean total RAAD score (p = 0.049). Conclusions: Patients with longstanding established RA have more severe irreversible joint damage as measured by the clinical RAAD score, contrary to other studies in Africa. This is largely reflected by a delay in the initiation of early effective treatment. Independent of disease duration, older age at disease onset and a higher RF titre are strongly associated with more joint damage. The inverse association between BMI and articular damage in RA has been observed in several studies using radiographic damage scores. The mechanisms underlying this paradoxical association are still widely unknown but adipokines have recently been suggested to play a role. Disclosure statement: C.I. has received a research grant from the Connective Tissue Diseases Research Fund, University of the Witwatersrand. All other authors have declared no conflicts of interes

Residues of the Rotavirus RNA-Dependent RNA Polymerase Template Entry Tunnel That Mediate RNA Recognition and Genome Replication▿

To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (+RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3′ end of rotavirus +RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral +RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with +RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of +RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with +RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates

Mutants used in this study.

<p>^aResidues are numbered by their codon position within each gene of the JFH-1 subgenomic replicon or in the polyprotein of HCV reference strain H77; n.a., not applicable.</p><p>Mutants used in this study.</p

Models of viral <i>cis</i>-activities and HCV replicase structure and function.

<p>(<b>A</b>) Ribosome-induced change in RNA structure. (<b>B</b>) Nascent <i>cis</i>-acting polypeptide (orange) recruiting the viral RNA-ribosome complex to a site of replication via an interaction partner (yellow). (<b>C</b>) A <i>cis</i>-acting protein (orange) recruiting the viral RNA to site of replication. (<b>D</b>) NS5B <i>trans</i>-complementation via protein transfer. (<b>E</b>) NS5B <i>trans</i>-complementation via RNA transfer. (<b>F</b>) NS5B <i>trans</i>-complementation requires NS3–5B expression. (<b>G</b>) Translation of NS5B is required in <i>cis</i>. (<b>H</b>) Potential NS3-NS4A interactions of the 3m6 and 4Am1 mutants. NS3-4A can form homodimers where each monomer contributes either NS3 or NS4A (left); alternatively, NS3-NS4A may dissociate and reassociate to form separate active and inactive monomers (right).</p

Complementation of NS3 serine protease and RNA helicase active site mutants.

<p>(<b>A</b>) Serine protease active site mutants SGR-Gluc(3m1) and SGR-Gluc(3m2) do not replicate and are not complemented in <i>trans</i>. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls. This experiment was performed two times with similar results. (<b>B</b>) Helicase RNA binding and NTPase active site mutants SGR-Gluc(3m3) and SGR-Gluc(3m4) do not replicate and are not complemented in <i>trans</i>. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls. For comparison, the SGR-Gluc samples, which were performed in parallel, are reproduced from panel A. This experiment was performed two times with similar results. (<b>C</b>) RNA helicase base stacking mutant SGR-Gluc(3m5) does not replicate but is complemented in <i>trans</i> with an efficiency similar to SGR-Gluc(5m1). For comparison, the SGR-Gluc samples, which were performed in parallel, are reproduced from panel A. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls; *, <i>p</i> < 0.05; **, <i>p</i> <0.01 by Student’s <i>t</i>-test, comparing matched time points from SGR-Gluc(3m5) in Huh-7.5[VEEV/NS3–5B] vs. Huh-7.5[VEEV/GFP] cells. This experiment was performed two times with similar results. (<b>D</b>) Western blot to confirm that the serine protease active site mutant SGR-Gluc(3m1) does not undergo polyprotein cleavage and that the RNA helicase mutants SGR-Gluc(3m2), SGR-Gluc(3m3), SGR-Gluc(3m4), and SGR-Gluc(3m5) do not exhibit defects in NS3 accumulation. NS3 was detected by using anti-NS3 monoclonal antibody 4E11; values represent relative NS3 levels compared to SGR-Gluc. Because these mutants do not replicate, expression was driven by the vaccinia-T7 RNA polymerase system. (<b>E</b>) Design of the Jc1-Gluc(3–5Arc) mutant. (<b>F</b>) Jc1-Gluc(3–5Arc) replicates efficiently. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls; n.s., <i>p</i> >0.05, *, <i>p</i> ≤ 0.05 by Student’s <i>t</i>-test. This experiment was performed two times with similar results (<b>G</b>) Model showing that SGR-Gluc(3m1) and SGR-Gluc(3m2) do not undergo proteolytic processing, produce misfolded, inactive polyproteins, and are not complemented in <i>trans</i>; grey signifies the nonfunctional serine protease domains; blue signifies the recoded NS3-5B polyprotein. (<b>H</b>) Model showing that SGR-Gluc(3m3) and SGR-Gluc(3m4) are not <i>trans</i>-complemented and may have a defect in RNA recruitment (compare to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004817#ppat.1004817.g001" target="_blank">Fig 1B</a>); grey signifies the nonfunctional helicase domains; blue signifies the recoded NS3-5B polyprotein.</p

A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry