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We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system. 相似文献
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Alanine scanning of the hepatitis C virus core protein reveals numerous residues essential for production of infectious virus 总被引:1,自引:0,他引:1
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Hepatitis C virus (HCV) is an important human pathogen affecting an estimated 3% of the world's population. Recent advances have enabled in vitro propagation of the virus and allow assembly and egress to be investigated for the first time. As a component of the virion, the HCV core protein likely functions primarily in infectious virus production, although little is known about the determinants of this activity. To investigate the roles of core in the viral life cycle, we performed a comprehensive deletion and alanine scanning mutagenesis study of this protein in the context of a genotype 2a reporter virus. We have confirmed that core protein is essential for infectious virion production and have identified numerous residues required for this role. The infectivity of several assembly-defective core mutants could be rescued by compensatory mutations identified in p7 and NS2, suggesting genetic interactions with core and highlighting the importance of these nonstructural proteins in infectious virion morphogenesis. 相似文献
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Hepatitis B virus core gene mutations which block nucleocapsid envelopment 总被引:9,自引:0,他引:9
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Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. 相似文献
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Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A
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Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex. 相似文献
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Temperature-Sensitive Mutants of Simian Virus 40: Infection of Permissive Cells 总被引:68,自引:44,他引:24
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Ten temperature-sensitive mutants of simian virus 40 have been isolated and characterized in permissive cells. The mutants could be divided into three functional groups and two complementation groups. Seven mutants produced T antigen, infectious viral deoxyribonucleic acid (DNA), and structural viral antigen but predominantly the empty shell type of viral particles. Two mutants produced T antigen and infectious viral DNA, but, although viral structural protein(s) could be detected immunologically, no V antigen or viral particles were found. These two functional groups of mutants did not complement each other. A single mutant was defective in the synthesis of viral DNA, viral structural antigens, and viral particles. T antigen could be detected in infected cells by fluorescent antibody but was reduced by complement fixation assay. This mutant stimulated cell DNA synthesis at the restrictive temperature and complemented the other two functional groups of mutants. 相似文献
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P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Correlation of Physical and Cytogenetic Maps in Chromosomal Region 86e-87f 总被引:2,自引:0,他引:2
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P. Deak M. M. Omar RDC. Saunders M. Pal O. Komonyi J. Szidonya P. Maroy Y. Zhang M. Ashburner P. Benos C. Savakis I. Siden-Kiamos C. Louis V. N. Bolshakov F. C. Kafatos E. Madueno J. Modolell D. M. Glover 《Genetics》1997,147(4):1697-1722
We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover ~25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven ``supercontigs' that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the αγ element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms. 相似文献
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Precise missense and silent point mutations are fixed in the genomes of poliovirus mutants from persistently infected cells. 总被引:12,自引:11,他引:1
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Poliovirus mutants selected in persistently infected human neuroblastoma cells have a modified cell tropism and can establish a secondary persistent infection in nonneural cells, such as HEp-2c cells. Nucleotide sequence analysis revealed that the genome of a persistent mutant, S11, differed from that of the parental lytic Sabin 1 poliovirus strain by 31 point mutations. Three mutations occurred in the noncoding regions. The other mutations resulted in 12 amino acid substitutions; 1 substitution occurred in a nonstructural protein (3A), while the other 11 substitutions were clustered in the capsid proteins VP2 and VP1. The same missense mutations, as well as many of the silent mutations that we observed in mutant S11, also accumulated in the genome of two other persistent viruses isolated from independent infections. This finding indicates that both missense and silent mutations are selected during the persistent infection of neuroblastoma cells and suggests that the secondary structure of RNA in the coding region may play a role in viral infection. 相似文献
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In flaviviruses it has been proposed that there is a coupling between genome replication and virion assembly and that nonstructural proteins are involved in this process. It was previously reported that mutations in yellow fever virus (YFV) nonstructural protein NS2A blocked production of infectious virus and that this block could be released by a suppressor mutation in NS3. Here, based on studies using a YFV replicon-based trans-packaging system as well as full-length YFV cDNA, we report that mutation of a conserved tryptophan at position 349 in the helicase domain of NS3 blocks production of infectious virus particles, revealing an as-yet-unknown role for NS3 in virus assembly. Mutation of tryptophan 349 to alanine (W349A) had no effect on viral replication, as demonstrated by wild-type levels of viral RNA amplification and protein expression in W349A-transfected cells. Although release of infectious virus was not detected, release of capsidless subviral particles was not blocked. The assembly defect in W349A could be trans-complemented inefficiently using BHK-REP cells (a cell line containing persistently replicating YFV replicon RNA). trans-complementation was also demonstrated by supplying wild-type NS2B-3 or NS3 protein alone as well as by supplying inactive NS2B-3 protein, indicating that this function of NS3 in virus assembly was independent of its known enzymatic functions. 相似文献
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Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome. 总被引:30,自引:24,他引:6
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V G Preston 《Journal of virology》1981,39(1):150-161
Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature. 相似文献
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L Willems R Kettmann F Dequiedt D Portetelle V Vonche I Cornil P Kerkhofs A Burny M Mammerickx 《Journal of virology》1993,67(7):4078-4085
Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine. 相似文献
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Genetic and phenotypic analysis of herpes simplex virus type 1 mutants conditionally resistant to immune cytolysis 总被引:15,自引:14,他引:1
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Nine temperature-sensitive (ts) mutants of herpes simplex virus type 1 selected for their inability to render cells susceptible to immune cytolysis after infection at the nonpermissive temperature have been characterized genetically and phenotypically. The mutations in four mutants were mapped physically by marker rescue and assigned to functional groups by complementation analysis. In an effort to determine the molecular basis for cytolysis resistance, cells infected with each of the nine mutants were monitored for the synthesis of viral glycoprotein in total cell extracts and for the presence of these glycoproteins in plasma membranes. The four mutants whose ts mutations were mapped were selected with polypeptide-specific antiserum to glycoproteins gA and gB; however, three of the four mutations mapped to DNA sequences outside the limits of the structural gene specifying these glycoproteins. Combined complementation and phenotypic analysis indicates that the fourth mutation also lies elsewhere. The ts mutations in five additional cytolysis-resistant mutants could not be rescued with single cloned DNA fragments representing the entire herpes simplex virus type 1 genome, suggesting that these mutants may possess multiple mutations. Complementation tests with the four mutants whose ts lesions had been mapped physically demonstrated that each represents a new viral gene. Examination of mutant-infected cells at the nonpermissive temperature for the presence of viral glycoproteins in total cell extracts and in membranes at the cell surface demonstrated that (i) none of the five major viral glycoproteins was detected in extracts of cells infected with one mutant, suggesting that this mutant is defective in a very early function; (ii) cells infected with six of the nine mutants exhibited greatly reduced levels of all the major viral glycoproteins at the infected cell surface, indicating that these mutants possess defects in the synthesis or processing of viral glycoproteins; and (iii) in cells infected with one mutant, all viral glycoproteins were precipitable at the surface of the infected cell, despite the resistance of these cells to cytolysis. This mutant is most likely mutated in a gene affecting a late stage in glycoprotein processing, leading to altered presentation of glycoproteins at the plasma membrane. The finding that the synthesis of both gB and gC was affected coordinately in cells infected with six of the nine mutants suggests that synthesis of these two glycoproteins, their transport to the cell surface, or their insertion into plasma membranes is coordinately regulated. 相似文献
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Toward a Molecular Genetic Analysis of Spermatogenesis in Drosophila Melanogaster: Characterization of Male-Sterile Mutants Generated by Single P Element Mutagenesis 总被引:12,自引:3,他引:9
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D. H. Castrillon P. Gonczy S. Alexander R. Rawson C. G. Eberhart S. Viswanathan S. DiNardo S. A. Wasserman 《Genetics》1993,135(2):489-505
We describe 83 recessive autosomal male-sterile mutations, generated by single P element mutagenesis in Drosophila melanogaster. Each mutation has been localized to a lettered subdivision of the polytene map. Reversion analyses, as well as complementation tests using available chromosomal deficiencies, indicate that the insertions are responsible for the mutant phenotypes. These mutations represent 63 complementation groups, 58 of which are required for spermatogenesis. Phenotypes of the spermatogenesis mutants were analyzed by light microscopy. Mutations in 12 loci affect germline proliferation, spermatocyte growth, or meiosis. Mutations in 46 other loci disrupt differentiation and maturation of spermatids into motile sperm. This collection of male-sterile mutants provides the basis for a molecular genetic analysis of spermatogenesis. 相似文献
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Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles
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Little is known about the function of flavivirus nonstructural protein NS2A. Two forms of NS2A are found in yellow fever virus-infected cells. Full-length NS2A (224 amino acids) is the product of cleavage at the NS1/2A and NS2A/2B sites. NS2Aalpha, a C-terminally truncated form of 190 amino acids, results from partial cleavage by the viral NS2B-3 serine protease at the sequence QK /T within NS2A. Exchange of serine for lysine at this site (QKT-->QST) blocks the production of both NS2Aalpha and infectious virus. The present study reveals that this defect is not at the level of RNA replication. Despite normal structural region processing, infectious particles containing genome RNA and capsid protein were not released from cells transfected with the mutant RNA. Nevertheless, production of subviral prM/M- and E-containing particles was unimpaired. The NS2A defect could be complemented in trans by providing NS1-2A or NS1-2Aalpha. However, trans complementation was not observed when the C-terminal lysine of NS1-2Aalpha was replaced with serine. In addition to true reversions, NS2Aalpha cleavage site mutations could be suppressed by two classes of second-site changes. The first class consisted of insertions at the NS2Aalpha cleavage site that restored its basic character and cleavability. A second class of suppressors occurred in the NS3 helicase domain, in which NS3 aspartate 343 was replaced with an uncharged residue (either valine, alanine, or glycine). These mutations in NS3 restored infectious-virus production in the absence of cleavage at the mutant NS2Aalpha site. Taken together, our results reveal an unexpected role for NS2A and NS3 in the assembly and/or release of infectious flavivirus particles. 相似文献
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Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C. 总被引:30,自引:24,他引:6
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Sequence analysis of the genomic RNA of interstrain guanidine-resistant and antibody-resistant variant recombinants of poliovirus type 1 mapped the resistance of mutants capable of growth in 2.0 mM guanidine hydrochloride to a region located 3' of nucleotide 4444. This region of the viral genome specifies the nonstructural protein 2C. The sequence of genomic RNA encoding 2C from six independently isolated mutants resistant to 2.0 mM guanidine was determined. All six isolates contained a mutation in 2C at the same position in all cases, resulting in two types of amino acid changes. Dependent mutants were examined and found to contain two amino acid changes each within 2C. Mutants resistant to 0.53 mM guanidine were isolated and found to lack the mutations seen in variants resistant to 2.0 mM guanidine. A comparison of the amino acid sequences of the 2C proteins of poliovirus, foot-and-mouth disease virus, rhinovirus types 2 and 14, and encephalomyocarditis virus revealed a strong homology over regions totaling 115 residues. All of the mutations observed in guanidine-selected mutants were contained within this region. The amino acid region containing the mutations observed in poliovirus mutants resistant to 2.0 mM guanidine was compared with the homologous region in the other picornaviruses; a strong correlation was found between the amino acid present at this position and the sensitivity of the virus to 2.0 mM guanidine. 相似文献
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Genetic analysis of the pestivirus nonstructural coding region: defects in the NS5A unit can be complemented in trans
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The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5' and 3' untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery. 相似文献