首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Replication of the picornavirus genome is catalysed by a viral encoded RNA-dependent RNA polymerase, termed 3D polymerase. Together with other viral and host proteins, this enzyme performs its functions in the cytoplasm of host cells. The crystal structure of 3D polymerase from a number of picornaviruses has been determined. This review discusses the structure and function of the poliovirus 3D polymerase. The high error rates of 3D polymerase result in high sequence diversity such that virus populations exist as quasispecies. This phenomenon is thought to facilitate survival of the virus population in complex environments.  相似文献   

2.
Influence of the biogenic polyamines spermine, spermidine, and putrescine as well as their derivatives on the replication enzymes of hepatitis C virus (HCV) was investigated. It was found that spermine and spermidine activate HCV RNA-dependent RNA polymerase (NS5B protein). This effect was not caused by the stabilization of the enzyme or by competition with template-primer complex, but rather it was due to achievement of true maximum velocity V max. Natural polyamines and their derivatives effectively inhibited the helicase reaction catalyzed by another enzyme of HCV replication — helicase/NTPase (NS3 protein). However, these compounds affected neither the NTPase reaction nor its activation by polynucleotides. Activation of the HCV RNA polymerase and inhibition of the viral helicase were shown at physiological concentrations of the polyamines. These data suggest that biogenic polyamines may cause differently directed effects on the replication of the HCV genome in an infected cell.  相似文献   

3.
The soluble phase of the cytoplasm of human rhinovirus type 2-infected cells contains an enzymatic activity able to copy rhinovirion RNA without an added primer. This RNA-dependent RNA polymerase (replicase) makes a specific copy of the added rhinovirion RNA, as shown by hybridization of the product to its template RNA but not to other RNAs. The same replicase preparation also contains a virus-specific polyuridylic acid [poly(U)] polymerase activity which is dependent on added polyadenylic acid-oligouridylic acid template-primer. Both activities purify together until a step at which poly(U) polymerase but no replicase activity is recovered. Addition of a purified HeLa cell protein (host factor) to this poly(U) polymerase completely reconstitutes rhinovirus replicase activity. Host factor activity can be supplied by adding oligouridylic acid, suggesting that the host cell protein acts at the initiation step of rhinovirus RNA replication. A virus-specific 64,000-dalton protein purifies with both poly(U) polymerase and replicase activities.  相似文献   

4.
Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.  相似文献   

5.
Replication of RNA by the DNA-dependent RNA polymerase of phage T7   总被引:11,自引:0,他引:11  
M M Konarska  P A Sharp 《Cell》1989,57(3):423-431
The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.  相似文献   

6.
7.
8.
9.
10.
11.
12.
13.
Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. These viruses also use a small protein primer, named VPg, to initiate RNA replication. The recent explosion of structural information on picornaviral 3D polymerases has provided insights into the initiation of RNA synthesis and chain elongation. Comparing these data with results from previous structural analyses of viral RNA-dependent RNA polymerases that catalyze de novo RNA synthesis sheds light on the different strategies that these viruses use to initiate replication.  相似文献   

14.
15.
16.
Two critical interactions within the poliovirus RNA replication complex are those of the RNA-dependent RNA polymerase 3D with the viral proteins 3AB and VPg. 3AB is a membrane-binding protein responsible for the localization of the polymerase to the membranous vesicles at which replication occurs. VPg (a peptide comprising the 3B region of 3AB) is the 22-residue soluble product of 3AB cleavage and serves as the protein primer for RNA replication. The detailed interactions of these proteins with the RNA-dependent RNA polymerase 3D were analyzed to elucidate the precise roles of 3AB and VPg in the viral RNA replication complex. Using a membrane-based pull-down assay, we have identified a binding "hot-spot" spanning residues 100 to 104 in the 3B (VPg) region of 3AB which plays a critical role in mediating the interaction of 3AB with the polymerase. Isothermal titration calorimetry shows that the interaction of VPg with 3D is enthalpically driven, with a dissociation constant of 11 microM. Mutational analyses of VPg indicate that a subset of the residues important for 3AB-3D binding are also important for VPg-3D binding. Two residues in particular, P14 and R17, were shown to be absolutely critical for the binding interaction. This work provides the direct characterization of two binding interactions critical for the replication of this important class of viruses and identifies a conserved polymerase binding sequence responsible for targeting the polymerase.  相似文献   

17.
R J Hayes  K W Buck 《Cell》1990,63(2):363-368
A soluble RNA-dependent RNA polymerase was isolated from Nicotiana tabacum plants infected with cucumber mosaic virus (CMV), which has a genome of three positive-strand RNA components, 1, 2, and 3. The purified polymerase contained two virus-encoded polypeptides and one host polypeptide. Polymerase activity was completely dependent on addition of CMV RNA as template, and the products of reaction were single-stranded (ss) RNA and double-stranded (ds) RNA, corresponding to RNAs 1, 2, and 3, and a subgenomic RNA (RNA 4) derived from RNA 3. The ratio of ssRNA to dsRNA was about 5:1, and the ssRNA was shown to be predominantly the positive strand. This demonstrates the complete replication of a eukaryotic virus RNA in vitro by a template-dependent RNA polymerase.  相似文献   

18.
IN spite of the apparent simplicity of RNA bacteriophage, several proteins, both phage and bacterial, are required for the synthesis of Qβ RNA in vitro. The polymerase complex alone contains one phage-coded and three host proteins1,2. The specific role of these proteins in Qβ RNA replication is unknown, but because they demonstrate an associative interaction and are always found with active enzyme, it has been suggested that all four contribute to polymerase activity1.  相似文献   

19.
In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug.  相似文献   

20.
M G Kramer  S A Khan    M Espinosa 《The EMBO journal》1997,16(18):5784-5795
Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号