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1.
The Caprine arthritis encephalitis virus (CAEV) vif gene was demonstrated to be essential for efficient virus replication. CAEV Vif deletion mutants demonstrated an attenuated replication phenotype in primary goat cell cultures and resulted in abortive infection when inoculated into goats. In this study, we determined the in vitro replication phenotype of five CAEV Vif point mutant infectious molecular clones and the ability of the corresponding in vitro translated Vif proteins to interact with the CAEV Pr55(gag) in the glutathione S--transferase (GST) binding assay. Here we show that (i) three of the mutants (S170E, S170G, S197G) behaved as the wild-type CAEV according to virus replication and Vif--Gag interactions; (ii) one mutant (Vif 6mut) was replication incompetent and bound weakly to GST-Gag fusion proteins; and (iii) one mutant (Vif RG) was impaired for replication while retaining its interaction properties. This mutant points out the critical importance of the CAEV Vif tryptophan residue at position 95 for efficient virus replication, defining for this lentivirus a functional domain unrelated to the Gag binding region. 相似文献
2.
Ravazzolo AP Nenci C Vogt HR Waldvogel A Obexer-Ruff G Peterhans E Bertoni G 《Virology》2006,350(1):116-127
Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed. 相似文献
3.
Cheevers WP Snekvik KR Trujillo JD Kumpula-McWhirter NM Pretty On Top KJ Knowles DP 《Virology》2003,306(1):116-125
This study evaluated the efficacy of prime-boost vaccination for immune control of caprine arthritis-encephalitis virus (CAEV), a macrophage tropic lentivirus that causes progressive arthritis in the natural host. Vaccination of Saanen goats with pUC-based plasmid DNA expressing CAEV env induces T helper type 1 (Th1) biased immune responses to vector-encoded surface envelope (SU), and the plasmid-primed Th1 response is expanded following boost with purified SU in Freund's incomplete adjuvant (SU-FIA) (J. C. Beyer et al., 2001, Vaccine 19, 1643-1651). Four goats vaccinated with env expression plasmids and boosted with SU-FIA were challenged intravenously with 1 x 10(4) TCID(50) of CAEV at 428 days after SU-FIA boost and evaluated by immunological, virological, and disease criteria. Controls included two goats primed with pUC18 and eight unvaccinated goats. Goats receiving prime-boost vaccination with CAEV env plasmids and SU-FIA became infected but suppressed postchallenge virus replication, provirus loads in lymph node, and development of arthritis for at least 84 weeks. 相似文献
4.
Suzuki R Saito K Kato T Shirakura M Akazawa D Ishii K Aizaki H Kanegae Y Matsuura Y Saito I Wakita T Suzuki T 《Virology》2012,432(1):29-38
In this study, we compared the entry processes of trans-complemented hepatitis C virus particles (HCVtcp), cell culture-produced HCV (HCVcc) and HCV pseudoparticles (HCVpp). Anti-CD81 antibody reduced the entry of HCVtcp and HCVcc to almost background levels, and that of HCVpp by approximately 50%. Apolipoprotein E-dependent infection was observed with HCVtcp and HCVcc, but not with HCVpp, suggesting that the HCVtcp system is more relevant as a model of HCV infection than HCVpp. We improved the productivity of HCVtcp by introducing adapted mutations and by deleting sequences not required for replication from the subgenomic replicon construct. Furthermore, blind passage of the HCVtcp in packaging cells resulted in a novel mutation in the NS3 region, N1586D, which contributed to assembly of infectious virus. These results demonstrate that our plasmid-based system for efficient production of HCVtcp is beneficial for studying HCV life cycles, particularly in viral assembly and infection. 相似文献
5.
Construction and applications of yellow fever virus replicons 总被引:10,自引:0,他引:10
Subgenomic replicons of yellow fever virus (YFV) were constructed to allow expression of heterologous reporter genes in a replication-dependent manner. Expression of the antibiotic resistance gene neomycin phosphotransferase II (Neo) from one of these YFV replicons allowed selection of a stable population of cells (BHK-REP cells) in which the YFV replicon persistently replicated. BHK-REP cells were successfully used to trans-complement replication-defective YFV replicons harboring large internal deletions within either the NS1 or NS3 proteins. Although replicons with large deletions in either NS1 or NS3 were trans-complemented in BHK-REP, replicons that contained deletions of NS3 were trans-complemented at lower levels. In addition, replicons that retained the N-terminal protease domain of NS3 in cis were trans-complemented with higher efficiency than replicons in which both the protease and helicase domains of NS3 were deleted. To study packaging of YFV replicons, Sindbis replicons were constructed that expressed the YFV structural proteins in trans. Using these Sindbis replicons, both replication-competent and trans-complemented, replication-defective YFV replicons could be packaged into pseudo-infectious particles (PIPs). Although these results eliminate a potential role of either NS1 or full-length NS3 in cis for packaging and assembly of the flavivirus virion, they do not preclude the possibility that these proteins may act in trans during these processes. 相似文献
6.
The requirement to replicate in both vertebrate and invertebrate hosts is thought to limit the introduction of genetic changes into the genome of arboviruses. Serial passage under laboratory conditions will overcome this limitation allowing for genetic changes to be introduced and affecting the virulence of the virus for animals. In the studies detailed here, the consequence of removing the restriction of alternate replication was demonstrated to be different depending on the virus. Passing Venezuelan equine encephalitis virus in tissue culture cells, eggs or mice resulted in up to 11 nucleotide or amino acid changes but no significant change in the virulence of the virus for mice. Passing Japanese encephalitis virus (JEV) under the identical conditions resulted in as many as 22 nucleotide or amino acid changes that often resulted in improved survival probabilities. For JEV, most genetic changes along with the attenuated phenotype were selected within 5 passes. 相似文献
7.
Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response 总被引:1,自引:0,他引:1
Bovine viral diarrhea virus (BVDV) replicons with deletions within the capsid, E(RNS) or E1 encoding region were constructed and efficiently packaged with a helper cell line. High titres of packaged replicons were observed as early as 24 h after transfection, whereas no virus progeny could be detected after transfection of non-complementing cells. Infection of bovine cell cultures with rescued viruses resulted in one cycle of replication without release of infectious virus particles, and no genetic reversion of the generated viruses was detected. Packaged replicons with a deletion within the capsid-coding region were characterized in vivo in immunization and challenge trials. Following immunization of calves with the replication-deficient virus, neither virus shedding nor viremia was detected. After challenge infection with virulent BVDV, all vaccinates were completely protected from disease as measured by the absence of viremia and shedding of challenge virus, which indicated that a 'sterilizing immunity' could be induced with the generated replication-deficient packaged replicons. 相似文献
8.
Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) belong to the Benyvirus genus. BSBMV has been reported only in the United States, while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae and possess similar host ranges, particle number and morphology. BNYVV and BSBMV are not serologically related but they have similar genomic organizations. Field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles in plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed that BSBMV and BNYVV are sufficiently different to be classified as two species. Complementary base changes found within the BSBMV RNA-3 5′ UTR made it resemble to BNYVV 5′ RNA-3 structure whereas the 3′ UTRs of both species were more conserved. cDNA clones were obtained, and allowed complete copies of BSBMV RNA-3 to be trans-replicated, trans-encapsidated by the BNYVV viral machinery. Long-distance movement was observed indicating that BSBMV RNA-3 could substitute BNYVV RNA-3 for systemic spread, even though the p29 encoded by BSBMV RNA-3 is much closer to the RNA-5-encoded p26 than to BNYVV RNA-3-encoded p25. Competition occurred when BSBMV RNA-3-derived replicons were used together with BNYVV-derived RNA-3 but not when the RNA-5-derived component was used. Exploitation of the similarities and divergences between BSBMV and BNYVV should lead to a better understanding of molecular interactions between Benyviruses and their hosts. 相似文献
9.
A selective trans-packaging system was developed to produce and isolate bovine viral diarrhea virus (BVDV) pseudo-particles with complementing reporter replicons and their packaging proteins expressed in trans with recombinant vaccinia virus. The encapsidation of replicon rNS3-5B was dependent not only on the in trans expression of structural proteins C, Erns, E1 and E2, but also the nonstructural proteins, p7 and contiguous precursor NS2-3-4A. Nonstructural p7, NS4B, NS5A or NS5B could be expressed in cis and in trans with precursor NS2-3-4A without significantly affecting virion assembly efficiency. NS2-3-4A was identified as an in trans functional precursor in virion assembly. BVDV genomes with mutant NS5B, which did not undergo active replication, were packaged 5-fold less efficiently than the intact genomes demonstrating the importance of replication in virion packaging. These results suggest that genome replication and assembly are closely associated, consistent with a model in which these two steps are coupled for maximum efficiency. 相似文献
10.
Western equine encephalitis virus (WEEV) is a positive-sense, single-stranded RNA virus which is transmitted to equines and humans through mosquito bites. WEEV infects the central nervous system with severe complications and even death. There are no human vaccine and antiviral drugs. We investigated whether adenovirus-mediated expression of interferon alpha could be used for pre- and post-exposure protection against a lethal WEEV challenge in mice. A human adenoviral vector (Ad5-mIFNalpha) expressing mouse interferon alpha was constructed. We found that Ad5-mIFNalpha provided 100% protection against various WEEV strains in mice after a single intramuscular inoculation at 24 h, 48 h or 1 week before the challenge. When given as a single inoculation at 6 h after the challenge, Ad5-mIFNalpha delayed the progress of WEEV infection and provided about 60% protection. Our findings suggest that adenovirus-mediated expression of interferon alpha can be an alternative approach for the prevention and treatment of WEEV infection. 相似文献
11.
Autonomously replicating Bovine viral diarrhea virus (BVDV) genomes (replicons) were constructed from the full-length BVDV cDNA clone pA/BVDV/Ins- (G. Meyers et al., J. Virol. 70, 8606-8613, 1996). The sequences coding for envelope protein E2, for E2 without the C-terminal transmembrane region, or for E2 and nonstructural protein p7 were deleted, and the resulting mutants were tested for their ability to replicate after transfection. All deletion mutants were able to replicate and to express the inserted green fluorescent protein but did not produce infectious progeny virus in bovine kidney PT cells. The replicons were also tested for their ability to be trans-complemented in the bovine cell line PT_805, which constitutively expresses BVDV structural proteins. E2-negative BVDV mutants were complemented and >10(6) infectious units were obtained at 24 h after transfection. Complementing PT_805 cells could only inefficiently be infected using trans-complemented virions, however, and low levels of virus production were observed when complemented BVDV was passaged using PT_805 cells. Similarly, infection of PT_805 cells with BVDV was highly inefficient, but transfection of full-length BVDV NCP7 RNA into PT_805 resulted in 10,000-fold higher virus titers when compared to those obtained 24 h after transfection of parental PT cells. We concluded that self-replicating E2-deleted BVDV RNAs can be efficiently trans-complemented by constitutively expressed E2, and that expression of BVDV structural proteins markedly influences susceptibility of cells to BVDV infection as well as BVDV titers after transfection of full-length BVDV RNA. 相似文献
12.
Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication 总被引:2,自引:0,他引:2
The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by approximately 37 and approximately 60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes. 相似文献
13.
Matthew B. Harms Janet Cady Craig Zaidman Paul Cooper Taha Bali Peggy Allred Carlos Cruchaga Michael Baughn Ryan T. Libby Alan Pestronk Alison Goate John Ravits Robert H. Baloh 《Neurobiology of aging》2013
Hexanucleotide repeat expansions in C9ORF72 are a common cause of familial and apparently sporadic amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The mechanism by which expansions cause neurodegeneration is unknown, but current evidence supports both loss-of-function and gain-of-function mechanisms. We used pooled next-generation sequencing of the C9ORF72 gene in 389 ALS patients to look for traditional loss-of-function mutations. Although rare variants were identified, none were likely to be pathogenic, suggesting that mutations other than the repeat expansion are not a common cause of ALS, and providing supportive evidence for a gain-of-function mechanism. We also show by repeat-primed PCR genotyping that the C9ORF72 expansion frequency varies by geographical region within the United States, with an unexpectedly high frequency in the Mid-West. Finally we also show evidence of somatic instability of the expansion size by Southern blot, with the largest expansions occurring in brain tissue. 相似文献
14.
López-Mejías R García-Bermúdez M González-Juanatey C Castañeda S Miranda-Filloy JA Gómez-Vaquero C Fernández-Gutiérrez B Balsa A Pascual-Salcedo D Blanco R González-Álvaro I Llorca J Martín J González-Gay MA 《Human immunology》2012,73(5):543-546
Rheumatoid arthritis (RA) is an inflammatory disease associated with accelerated atherosclerosis. CXCL12 is a strong chemotactic signal for lymphocytes. Because previous genome-wide association studies demonstrated an association between CXCL12 rs501120 and coronary artery disease, in the present study we assessed the potential association of this polymorphism with the risk of cardiovascular (CV) disease in 1,321 Spanish patients with RA. A subgroup of patients without CV events was also studied to determine the presence of subclinical atherosclerosis by ultrasonography (brachial flow-mediated endothelium-dependent vasodilatation and carotid intima-media wall thickness). However, no significant differences in genotypic and allelic frequencies between RA patients with and without CV events were observed, as was also the case when values of surrogate markers of atherosclerosis were assessed according to CXCL12 rs501120 genotype frequencies. In conclusion, our results do not confirm an association of the CXCL12 rs501120 polymorphism with atherosclerosis or with CV disease in RA. 相似文献
15.
RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP-CP interaction occurs. 相似文献
16.
The genome of Maize chlorotic dwarf virus (MCDV; genus Waikavirus; family Sequiviridae) consists of a monopartite positive-sense RNA genome encoding a single large polyprotein. Antibodies were produced to His-fusions of three undefined regions of the MCDV polyprotein: the N-terminus of the polyprotein (R78), a region between coat proteins (CPs) and the nucleotide-binding site (NBS) (R37), and a region between the NBS and a 3C-like protease (R69). The R78 antibodies react with proteins of 50 kDa (P50), 35 kDa (P35), and 25 kDa (P25) in virus preparations, and with P35 in plant extracts. In extracts of the leafhopper vector Graminella nigrifrons fed on MCDV-infected plants, the R78 antibodies reacted with P25 but not with P50 and P35. The R69 antibodies bound proteins of approximately 36 kDa (P36), 30 kDa (P30), and 26 kDa (P26) in virus preparations, and P36 and P26 in plant extracts. Antibodies to R37 reacted with a 26-kDa protein in purified virus preparations, but not in plant extracts. Neither the R69 nor the R37 antibodies bound any proteins in G. nigrifrons. Thus, in addition to the three CPs, cysteine protease and RNA-dependent RNA polymerase, the MCDV polyprotein is apparently post-transitionally cleaved into P50, P35, P25, P36, P30, and P26. 相似文献
17.
B. I. Islamov V. A. Funtikov R. V. Bobrovskii Yu. V. Gotovskii 《Bulletin of experimental biology and medicine》1999,128(5):1112-1115
Bioresonance therapy normalized protein synthesis in lymphocytes inhibited by 60% in patients with rheumatoid arthritis, restored
impaired synthesis of heat shock proteins at rest (73 and 65 kD proteins) and after heat shock induction (120, 87, 73 and
72, 65, 55, 32 kD, respectively), and provided high induction level for 70 and 32 kD proteins compared to healthy subjects.
It is assumed that therapeutic effect of bioresonance therapy is partially determined by recovery of functional activity of
lymphocytes due to normalization of heat shock protein synthesis.
Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 11, pp. 525–528, November, 1999 相似文献
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De Buck E Höper D Lammertyn E Hecker M Anné J 《International journal of medical microbiology : IJMM》2008,298(5-6):449-461
The twin-arginine translocation (Tat) pathway is a secretory pathway for translocation of folded proteins with two arginines in their signal peptide across the cytoplasmic membrane. Recently, we showed the presence of the Tat secretion pathway in Legionella pneumophila Philadelphia-1 and its role in intracellular replication and biofilm formation. To analyse the importance of the Tat pathway in protein export and its role in L. pneumophila virulence, a comparative 2-D protein gel electrophoresis analysis was performed on supernatants of the wild type and two Tat secretion mutants in order to identify possible Tat substrates. Twenty proteins were identified as differential proteins, eight of which were present in a lower quantity in the supernatant of the tat mutants. Among these, one protein with a typical twin-arginine motif in its signal peptide was identified as the 3′,5′-cyclic nucleotide phosphodiesterase. Two other proteins that resulted as differential proteins from this study were flagellin and LvrE, which were studied in more detail and their Tat-dependence was further confirmed with specific antibodies. LvrE was shown to play a role in intracellular growth in differentiated U937 cells. 相似文献