Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.
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PMID:Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro. 1903 96

Hepatitis E virus (HEV) ORF1 protein (pORF1) contains methyltransferase (MetT), papain-like cysteine protease (PCP), RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. ORF1 sequence analysis showed two consensus LXGG cleavage sites at 664 and 1205. LXGG sequence is recognized by viral and cellular deubiquitinating enzymes. The protein encompassing the predicted MetT-PCP domains of HEV ORF1 was tested for deubiquitinating activity using fluorogenic substrates - ubiquitin-7-amino-4-methylcoumarin (AMC), IFN-stimulated gene 15 (ISG15)-AMC, Nedd8-AMC and SUMO-AMC. MetT-PCP cleaved all four substrates but processing of ISG15-AMC was more robust. There was no processing of the Hel and RdRp domains having the conserved (1205) LXGG site by the protein. MetT-PCP carried out deISGylation of the ISG15-conjugated cellular proteins, suggesting a possible role in combating cellular antiviral pathways.
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PMID:Deubiquitination activity associated with hepatitis E virus putative papain-like cysteine protease. 2165 54

Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication.
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PMID:SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication. 2763 Feb 38

Sumoylation is a transient, reversible dynamic posttranslational modification that regulates diverse cellular processes including plant-pathogen interactions. Sumoylation of NPR1, a master regulator of basal and systemic acquired resistance to a broad spectrum of plant pathogens, activates the defense response. Here, we report that NIb, the only RNA-dependent RNA polymerase of Turnip mosaic virus (TuMV) that targets the nucleus upon translation, interacts exclusively with and is sumoylated by SUMO3 (SMALL UBIQUITIN-LIKE MODIFIER3), but not the three other Arabidopsis thaliana SUMO paralogs. TuMV infection upregulates SUMO3 expression, and the sumoylation of NIb by SUMO3 regulates the nuclear-cytoplasmic partitioning of NIb. We identified the SUMO-interacting motif in NIb that is essential for its sumoylation and found that knockout or overexpression of SUMO3 suppresses TuMV replication and attenuates viral symptoms, suggesting that SUMO3 plays dual roles as a host factor of TuMV and as an antiviral defender. Sumoylation of NIb by SUMO3 is crucial for its role in suppressing the host immune response. Taken together, our findings reveal that sumoylation of NIb promotes TuMV infection by retargeting NIb from the nucleus to the cytoplasm where viral replication takes place and by suppressing host antiviral responses through counteracting the TuMV infection-induced, SUMO3-activated, NPR1-mediated resistance pathway.
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PMID:Sumoylation of Turnip mosaic virus RNA Polymerase Promotes Viral Infection by Counteracting the Host NPR1-Mediated Immune Response. 2822 39

SUMOylation is a posttranslational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. In this study, we found that the VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that residues ⁴⁰⁴I and ⁴⁰⁶I within SUMO interaction motif 3 of VP1 constitute the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. A reverse genetic approach showed that only IBDV with I⁴⁰⁴C/T and I⁴⁰⁶C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.IMPORTANCE SUMOylation is an extensively discussed posttranslational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation comprised residues ⁴⁰⁴I and ⁴⁰⁶I of SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation-deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.
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PMID:SUMO1 Modification Facilitates Avibirnavirus Replication by Stabilizing Polymerase VP1. 3084 28

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