Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.
 ...
PMID:Identification of mouse hepatitis virus papain-like proteinase 2 activity. 1093 99

Mouse hepatitis virus (MHV) RNA synthesis is mediated by a viral RNA-dependent RNA polymerase (RdRp) on membrane-bound replication complexes in the host cell cytoplasm. However, it is not known how the putative MHV RdRp (Pol) is targeted to and retained on cellular membranes. In this report, we show that a 100-kDa protein was stably detected by an anti-Pol antiserum as a mature product throughout the virus life cycle. Gradient fractionation and biochemical extraction experiments demonstrated that Pol was not an integral membrane protein but was tightly associated with membranes and coimmunoprecipitated with the replicase proteins 3CLpro, p22, and p12. By immunofluorescence confocal microscopy, Pol colocalized with viral proteins at replication complexes, distinct from sites of virion assembly, over the entire course of infection. To determine if Pol associated with cellular membranes in the absence of other viral factors, the pol domain of gene 1 was cloned and expressed in cells as a fusion with green fluorescent protein, termed Gpol. In Gpol-expressing cells that were infected with MHV, but not in mock-infected cells, Gpol relocalized from a diffuse distribution in the cytoplasm to punctate foci that colocalized with markers for replication complexes. Expression of Gpol deletion mutants established that the conserved enzymatic domains of Pol were dispensable for replication complex association, but a 38-amino-acid domain in the RdRp unique region of Pol was required. This study demonstrates that viral or virus-induced factors are necessary for Pol to associate with membranes of replication complexes, and it identifies a defined region of Pol that may mediate its interactions with those factors.
 ...
PMID:Characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus RNA-dependent RNA polymerase. 1297 Apr 36

The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (non-structural proteins nsp1-nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12-nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II-VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicase-transcriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the non-permissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive- and negative-strand RNA. On the basis of these results, we propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved.
 ...
PMID:Functional and genetic analysis of coronavirus replicase-transcriptase proteins. 1634 Dec 54