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At least 217 records · Page 12Linked to original sources
PubMed1995

Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides.

A set of mutations [Ala-62-->Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers on the virus a reduced sensitivity to multiple antiretroviral dideoxynucleosides and has been seen in HIV-1 variants isolated from patients receiving combination chemotherapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddI). The IC50 values of AZT, ddC, ddI, 2',3'-dideoxyguanosine, and 2',3'-didehydro-3'-deoxythymidine against an infectious clone constructed to include the five mutations were significantly higher than those of a wild-type infectious clone. The K1 value for AZT 5'-triphosphate determined for the virus-associated RT from a posttherapy strain was 35-fold higher than that of RT from a pretherapy strain. Detailed analysis of HIV-1 strains isolated at various times during therapy showed that the Q151M mutation developed first in vivo, at the time when the viremia level suddenly increased, followed by the F116Y and F77L mutations. All five mutations ultimately developed, and the viremia level rose even further. Analyses based on the three-dimensional structure of HIV-1 RT suggest that the positions where at least several of the five mutations occur are located in close proximity to the proposed dNTP-binding site of RT and the first nucleotide position of the single-stranded template.

Acquired Immunodeficiency Syndrome↗
PubMed1981

Molecular cloning of infectious DNA from human papovavirus BK in Escherichia coli.

Recombinant DNA constructed from unit length BK virus DNA and from several defective viral forms was cloned in Escherichia coli HB101. The cloned unit length BK virus DNA retained its infectivity for human embryonic kidney cells, whereas the cloned defective DNA showed no infectivity. Restriction endonuclease digestion of cloned defective DNA and purified virus DNA indicated that many of the defective forms contain re-iterated sequences.

Cloning, Molecular↗
PubMed2006

Recovery of a neurovirulent human coronavirus OC43 from an infectious cDNA clone.

This study describes the assembly of a full-length cDNA clone of human coronavirus (HCoV)-OC43 in a bacterial artificial chromosome (BAC). The BAC containing the full-length infectious cDNA (pBAC-OC43(FL)) was assembled using a two-part strategy. The first step consisted in the introduction of each end of the viral genome into the BAC with accessory sequences allowing proper transcription. The second step consisted in the insertion of the whole HCoV-OC43 cDNA genome into the BAC. To produce recombinant viral particles, pBAC-OC43(FL) was transfected into BHK-21 cells. Recombinant virus displayed the same phenotypic properties as the wild-type virus, including infectious virus titers produced in cell culture and neurovirulence in mice.

Animals↗
PubMed2003

Deletions in the putative cell receptor-binding domain of Sindbis virus strain MRE16 E2 glycoprotein reduce midgut infectivity in Aedes aegypti.

The Sindbis virus (Alphavirus; Togaviridae) strain MRE16 efficiently infects Aedes aegypti mosquitoes that ingest a blood meal containing 8 to 9 log(10) PFU of virus/ml. However, a small-plaque variant of this virus, MRE16sp, poorly infects mosquitoes after oral infection with an equivalent titer. To determine the genetic differences between MRE16 and MRE16sp viruses, we have sequenced the MRE16sp structural genes and found a 90-nucleotide deletion in the E2 glycoprotein that spans the 3' end of the coding region for the putative cell-receptor binding domain (CRBD). We examined the role of this deletion in oral infection of mosquitoes by constructing infectious clones pMRE16icDeltaE200-Y229 and pMRE16ic, representing MRE16 virus genomes with and without the deletion, respectively. A third infectious clone, pMRE16icDeltaE200-C220, was also constructed that contained a smaller deletion extending only to the 3' terminus of the CRBD coding region. Virus derived from pMRE16ic replicated with the same efficiency as parental virus in vertebrate (BHK-21) and mosquito (C6/36) cells and orally infected A. aegypti. Viruses derived from pMRE16icDeltaE200-Y229 and pMRE16icDeltaE200-C220 replicated 10- to 100-fold less efficiently in C6/36 and BHK-21 cells than did MRE16ic virus. Each deletion mutant poorly infected A. aegypti and dramatically reduced midgut infectivity and dissemination. However, all viruses generated nearly equal titers (approximately 6.0 log(10) PFU/ml) in mosquitoes 4 days after infection by intrathoracic inoculation. These results suggest that the deleted portion of the E2 CRBD represents an important determinant of MRE16 virus midgut infectivity in A. aegypti.

Aedes↗
PubMed2006

Determinants of vector specificity of o'nyong nyong and chikungunya viruses in Anopheles and Aedes mosquitoes.

The alphaviruses o'nyong nyong virus (ONNV) and chikungunya virus (CHIKV) provide a unique system to study the viral genes involved in vector specificity. ONNV infects both anopheline and culicine mosquitoes, whereas CHIKV infects only culicine mosquitoes. In this study, chimeric viruses were constructed that contained genes from both ONNV and CHIKV. These chimeras and previously described full-length infectious clones of ONNV and CHIKV were evaluated in Anopheles gambiae and Aedes aegypti mosquitoes. Virus derived from the infectious clones of ONNV and CHIKV retained the vector specificity of the parental viruses. All six of the chimeras were found to infect Ae. aegypti mosquitoes at high rates but only the chimera containing viral genes encoding all of the structural proteins of ONNV was able to infect An. gambiae mosquitoes. These data indicate that all of the viral structural proteins are necessary for ONNV to infect An. gambiae mosquitoes.

Aedes↗
PubMed2002

Mapping the rubella virus subgenomic promoter.

Rubella virus (RUB), the sole member of the Rubivirus genus in the Togaviridae family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3' end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5'-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) -175 to +76 relative to the SG start site, including the 3' 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5' Deletions of SGP-2 to nt -40 (9 nt beyond the 3' end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5' deletions to nt -26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt -28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt -28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5' end of SG RNA was also required. Thus, the minimal SGP maps from nt -26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus Alphavirus, the other genus in the Togaviridae family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt -48 and nt -23 with respect to the SG start site in the RUB genome.

Animals↗
PubMed1985

In vitro mutagenesis of infectious viroid cDNA clone.

Previously we constructed an infectious hop stunt viroid (HSV) cDNA clone, PHS-P2P, which carries two copies of full-length HSV cDNA tandemly and generates HSV RNA when it is inoculated into cucumber plants. The in vitro transcript of the cDNA clone was also infectious. To investigate the essential regions for infectivity of HSV, we introduced a short deletion or insertion into the HSV sequence of pHS-P2P using restriction sites (XhoI site for pHI-X1, PvuI site for pHI-P1, and BamHI site for pHI-B1) and assayed the infectivities of these mutagenized clones. None of these mutagenized clones and their transcripts were infectious under the conditions used. Simultaneous inoculation of two or three of non-infectious mutagenized clones or the transcripts from them did not restore the infectivity.

Base Sequence↗
PubMed1997

In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

Animals↗
PubMed2003

Escherichia coli gets a new virus but it's nothing to sneeze at.

For the past two decades virologists have strived to make full-length clones of viral genomes that, on transfection into permissive eukaryotic cells, initiate a productive infection. The large variety of viral RNA and DNA genome structures, as well as different replication strategies, has required investigators to develop new approaches to produce infectious DNA in Escherichia coli. A member of the poxviridae, one of the most complex virus families, has now been made into an infectious clone in E. coli for the first time. Although the isolation was complicated, the infectious clone will greatly simplify future genetic studies of the virus.

Cloning, Molecular↗
PubMed1995

Sequence of host-range determinants in the env gene of a full-length, infectious proviral clone of exogenous avian leukosis virus HPRS-103 confirms that it represents a new subgroup (designated J).

A genomic DNA library was constructed, in a bacteriophage lambda vector, from line 0 chick embryo fibroblasts (CEFs) infected with HPRS-103, an exogenous avian leukosis virus (ALV; envelope subgroup J) recently isolated from meat-type chickens. The library was screened at high stringency using a full length RAV-1 (subgroup A) proviral probe. From 10(6) plaques, two clones which hybridized strongly to the RAV-1 probe were isolated; one contained a full-length copy of the proviral genome of HPRS-103 and the other contained a copy lacking the 5'-long terminal repeat (LTR) and part of gag. The relative strength of hybridization of RAV-1 and HPRS-103 clones, to RAV-1 probes representing different parts of the proviral genome, indicated that the gag and pol genes of HPRS-103 share a high level of identity with those of RAV-1 but that the env gene and the LTRs are considerably less well conserved. Infectious virus was recovered from CEFs transfected with the full-length clone, as detected by ELISA. The recovered virus appeared to be identical to HPRS-103 by electron microscopy and by Southern blotting of proviral DNA. The recovered virus was shown to be of the same subgroup as HPRS-103 by serum neutralization and receptor interference assays. Sequence analysis of the env gene of HPRS-103 shows that it differs considerably from the env genes of other ALV subgroups, particularly in the host range determinants, consistent with the finding that HPRS-103 represents a new subgroup (designated J).

Amino Acid Sequence↗
PubMed1994

The complete nucleotide sequence and construction of an infectious cDNA clone of a highly virulent encephalomyocarditis virus.

The nucleotide sequence of a highly variant of the encephalomyocarditis virus, PV21, was determined. Discounting the poly(A) tail the viral genome is 7.861 kb in length, including a poly(C) region of 157 or 158 nucleotides. The sequence differs in 38 positions from another virulent EMCV variant, EMCV-R (1). However, the PV21 sequence shows only 85% identity with two nonlethal variants isolated in this laboratory (2). A cDNA clone covering the complete virus sequence including a poly(C) region of approximately 150 nucleotides was constructed. The recombinant virus was shown to be infectious in NMRI mice.

Amino Acid Sequence↗
PubMed2003

Functional analysis of the grapevine virus A genome.

Grapevine virus A (GVA) carries five open reading frames (ORFs). Only the coat protein ORF has been experimentally identified as such; the roles of some of the other ORFs have been deduced by sequence homology to known genes (Minafra et al., 1997). The construction of a full-length, infectious clone of GVA has been previously reported. In an attempt to experimentally define the role of the various genes of GVA, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. Mutations in ORF 1 abolished RNA replication. Mutations in ORF 2 did not affect any of the aforementioned parameters. Mutations in ORFs 3 and 4 restricted viral movement. Mutations in ORF 5 rendered the virus asymptomatic, and partially restricted its movement.

Capsid Proteins↗
PubMed1995

In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease.

Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.

Amino Acid Sequence↗
PubMed2003

Frameshift mutations in infectious cDNA clones of Citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to Escherichia coli.

The advent of reverse genetics revolutionized the study of positive-stranded RNA viruses that were amenable for cloning as cDNAs into high-copy-number plasmids of Escherichia coli. However, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to E. coli. We report a strategy that is a compromise between infectivity of the RNA transcripts and toxicity to E. coli effected by introducing frameshift mutations into "slippery sequences" near the viral "toxicity sequences" in the viral cDNA. Citrus tristeza virus (CTV) has cDNA sequences that are toxic to E. coli. The original full-length infectious cDNA of CTV and a derivative replicon, CTV-DeltaCla, cloned into pUC119, resulted in unusually limited E. coli growth. However, upon sequencing of these cDNAs, an additional uridinylate (U) was found in a stretch of U's between nts 3726 and 3731 that resulted in a change to a reading frame with a stop codon at nt 3734. Yet, in vitro produced RNA transcripts from these clones infected protoplasts, and the resulting progeny virus was repaired. Correction of the frameshift mutation in the CTV cDNA constructs resulted in increased infectivity of in vitro produced RNA transcripts, but also caused a substantial increase of toxicity to E. coli, now requiring 3 days to develop visible colonies. Frameshift mutations created in sequences not suspected to facilitate reading frame shifting and silent mutations introduced into oligo(U) regions resulted in complete loss of infectivity, suggesting that the oligo(U) region facilitated the repair of the frameshift mutation. Additional frameshift mutations introduced into other oligo(U) regions also resulted in transcripts with reduced infectivity similarly to the original clones with the +1 insertion. However, only the frameshift mutations introduced into oligo(U) regions that were near and before the toxicity region improved growth and stability in E. coli. These data demonstrate that, when hosts are sufficiently susceptible for infection by transcripts of reduced specific infectivity, introduction of frameshift mutations at "slippery sequences" near toxic regions of viral cDNAs can be used as an additional strategy to clone recalcitrant viral sequences in high-copy-number plasmids for reverse genetics.

Citrus↗
PubMed2006

Combined amino acid mutations occurring in the envelope closely correlate with pathogenicity of EIAV.

The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system to study the attenuation mechanism and immunological control of lentivirus replication. Critical consensus mutations were identified between virulent Chinese EIAV strains and vaccine strains. Based on a full-length infectious clone of EIAV vaccine strain pLGFD3, two molecular clones, mFD5-4-7 and mFD7-2-11, were successfully constructed, in which 4 and 6 critical consensus mutations in the env gene of the vaccine strain were point-mutated to the wild-type sequence, respectively by an overlap PCR mutagenesis strategy. The infectivity, virulence, and pathogenesis of the constructed clones were investigated in vitro using a reverse transcriptase assay, an indirect immunofluorescence assay, observation of cytopathogenic effect, and virion observation as well as in vivo by inoculation of animals with the resulting infectious clones. The pathogenic symptoms in horses inoculated with mFD7-2-11 were more severe than those inoculated with mFD5-4-7, whereas no pathogenic symptoms were detected in animals inoculated with their parental clone pLGFD3 strain. The results indicate that the consensus mutation residues of the env region involved in this study play significant roles in the virulence and pathogenicity of EIAV. This will contribute to the elucidation of the attenuating and protective mechanisms of the Chinese EIAV vaccine.

Amino Acid Sequence↗
PubMed2004

Generation of an infectious cDNA clone of an FMDV strain isolated from swine.

A full-length cDNA clone of a foot-and-mouth disease virus (FMDV) isolated from swine was assembled in, the plasmid vector pBluescript II SK+ downstream of a T7 promoter. RNA synthesized in vitro using T7 polymerase lead to the production of infectious particles upon transfection of BHK-21 cells, as shown by cytopathic effects. The rescued virus was also found to be highly pathogenic for mice by intradermal injection producing a fatal disease indistinguishable from that of wild-type virus. The availability of this cDNA clone will allow examination of the molecular mechanisms behind FMDV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered FMDV vaccines.

Animals↗
PubMed2006

Manipulation of the yellow fever virus non-structural genes 2A and 4B and the 3'non-coding region to evaluate genetic determinants of viral dissemination from the Aedes aegypti midgut.

Although much is known about the ecology, epidemiology, and molecular biology of mosquito-borne viruses, the viral factors that allow transmission by mosquitoes to humans or animals remain unknown. Using infectious clones of disseminating (Asibi) and non-disseminating (17D) yellow fever viruses (YFV), we produced chimeric viruses to evaluate the role of different viral genes in dissemination. Previously, we showed that virus produced from an infectious clone containing the structural genes of 17D in Asibi disseminated from the mosquito midgut at a rate of 31%, indicating that some genetic determinants of dissemination must lie within the non-structural (NS) protein genes or 3' non-coding region (NCR). We chose to investigate the roles of NS2A, NS4B, and the 3'NCR in YFV dissemination. Substitution of the 17D NS2A or NS4B into Asibi significantly attenuated YFV dissemination, demonstrating that this is a multigenic property. There was no difference in dissemination after substitution of the 17D 3'NCR.

Aedes↗