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At least 235 records · Page 13Linked to original sources
PubMed2003

Analysis of mutant NS5B proteins encoded by isolates from chimpanzees chronically infected following clonal HCV RNA inoculation.

We hypothesized that mutations in the HCV NS5B polymerase, which occur during infection, may affect RNA-dependent RNA polymerase (RdRp) activity. NS5B proteins corresponding to a genotype 1a infectious clone and mutants identified in chimpanzees following inoculation with the clone were expressed and purified and their in vitro RdRp activity was compared to a NS5B genotype 1b control. A Gln-65-to-His mutation increased RdRp activity by 1.8-fold as compared to the infectious clone. Moreover, this NS5B1a protein had RdRp activity similar to the NS5B1b control. Three NS5B proteins representing mutations found in another animal had no in vitro RdRp activity. All mutations were maintained in the majority circulating virus for at least 216 weeks. The results demonstrate that some in vivo mutations of NS5B directly enhance in vitro RdRp activity. In addition, they suggest that the in vitro RdRp activity of NS5B may not always reflect in vivo activity within replication complexes.

Amino Acid Sequence↗
PubMed1993

Construction of an infectious DNA clone of the Y1 strain of canine parvovirus and characterization of the virus derived from the clone.

We have cloned genome fragments of canine parvovirus strain Y1 from replicative-form DNA and double-stranded DNA synthesized from virion DNA in vitro, and constructed a recombinant plasmid containing a full-length Y1 genome (pCPVY 1). When this recombinant plasmid was transfected into cell cultures, an infectious virus could be recovered. To characterize this pCPVY 1-derived virus, its biological properties were compared with those of the parental strain. No difference was observed between them in antigen expression, viral DNA replication, hemagglutination ability, and virus multiplication, indicating that the virus derived from the infectious plasmid inherited the biological properties of the authentic Y1 strain. Therefore, this recombinant plasmid appears to be useful for reverse genetics of canine parvovirus.

Animals↗
PubMed1991

Rice tungro bacilliform virus DNA independently infects rice after Agrobacterium-mediated transfer.

In nature, rice tungro disease is caused by an RNA and a DNA virus complex, but we have obtained an independently infectious clone of rice tungro bacilliform virus (RTBV) DNA. Infectivity could be demonstrated only when a more than unit-length copy was cloned in the Agrobacterium binary vector Bin 19 and agroinoculated into rice plants. Rice plants thus agroinfected with cloned RTBV DNA showed typical symptoms of tungro disease, presence of viral DNA and bacilliform particles, and could be used as a source of virus to infect healthy plants by the green leafhopper (Nephotettix virescens). The importance of this infectious clone in understanding the molecular biology of RTBV and the rice tungro disease is discussed.

DNA, Viral↗
PubMed2004

[The reverse genetics systems for human and animal RNA viruses].

The recovery of the virus from genetic materials in in vitro culture systems or sensitive animals is called virus rescue. A functional infectious clone of RNA virus provides unlimited possibility for genetic studies and the related reverse genetics system that allows directed genetic manipulation of an RNA virus is an extremely powerful research tool. In the past twenty years, especially since the first infectious clone of a negative-stranded RNA virus was reported in the mid-1990's, the reverse genetics systems have been available for nearly all the major human and animal RNA virus groups. The article reviews the progress of this technology, highlighting the obstacles in the construction of reverse genetics systems for major groups of human as well as animal RNA viruses and how the virologists overcame them. There are mainly four external expression systems for construction of the RNA virus reverse genetics systems basing on the kind of RNA viruses. These systems include in vitro RNA transcripts, RNA polymerase I-driven expression plasmids, RNA polymerase II-driven expression plasmids and modified vaccinia virus/T7 RNA polymerase-driven expression system. In particular, the viral nucleoprotein and polymerase proteins are required to assemble the viral ribonucleoprotein (RNP) complexes for the rescue of the negative-stranded RNA viruses. Relevant topics about the rescue of the typical viruses are discussed, including poliovirus with the de novo synthesis, Coronaviridae with the largest size of genome, Flaviviridae with the instable clones, HCV with the quasispecies nature, nodaviruses with the virus-host interaction, influenza virus with the RNA pol I transcription system, Arenavirdae with the ambisense coding strategies etc.

Animals↗
PubMed1988

Inability of Kaplan radiation leukemia virus to replicate on mouse fibroblasts is conferred by its long terminal repeat.

The molecularly cloned infectious Kaplan radiation leukemia virus has previously been shown to be unable to replicate on mouse fibroblasts (E. Rassart, M. Shang, Y. Boie, and P. Jolicoeur, J. Virol. 58:96-106, 1986). To map the viral sequences responsible for this, we constructed chimeric viral DNA genomes in vitro with parental cloned infectious viral DNAs from the nonfibrotropic (F-) BL/VL3 V-13 radiation leukemia virus and the fibrotropic (F+) endogenous BALB/c or Moloney murine leukemia viruses (MuLV). Infectious chimeric MuLVs, recovered after transfection of Ti-6 lymphocytes with these recombinant DNAs, were tested for capacity to replicate on mouse fibroblasts in vitro. We found that chimeric MuLVs harboring the long terminal repeat (LTR) of a fibrotropic MuLV replicated well on mouse fibroblasts. Conversely, chimeric MuLVs harboring the LTR of a nonfibrotropic MuLV were restricted on mouse fibroblasts. These results indicate that the LTR of BL/VL3 radiation leukemia virus harbors the primary determinant responsible for its inability to replicate on mouse fibroblasts in vitro. Our results also show that the primary determinant allowing F+ MuLVs (endogenous BALB/c and Moloney MuLVs) to replicate on mouse fibroblasts in vitro resides within the LTR.

Animals↗
PubMed2002

Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron.

The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.

Animals↗
PubMed1997

Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus.

The nonstructural region of the genome of the avirulent A7 strain of Semliki Forest virus (SFV) has been sequenced, so that the complete nucleotide sequence is available. Compared to the virulent SFV4 strain (produced from the infectious clone pSP6-SFV4), A7 contains 226 nucleotide changes in the translated region, which result in 47 amino acid changes. The 5' nontranslated region has two nucleotide changes, and the 3' nontranslated region is longer in A7 than SFV4, and contains divergent and repeated sequences. Chimeras containing SFV4 and A7 sequences and an infectious clone of A7, pSP6-CA7, were constructed. The virulence of these was tested by intraperitoneal and intranasal infection of adult BALB/c mice. It was shown that determination of the avirulent phenotype of A7 was polygenic, and required the additive effect of sequences from both the structural and non-structural regions of the SFV genome.

Animals↗
PubMed1993

Molecular cloning of infectious ecotropic murine leukemia virus AK7 from an emv-14-positive AKXL-5 mouse and the resistance of AK7 to recognition by cytotoxic T lymphocytes.

The AKXL-5 recombinant inbred mouse strain is positive for the endogenous ecotropic murine leukemia virus emv-14, the only emv present in its germ line. emv-14 is of particular interest because spleen cells expressing emv-14 virus escape recognition by anti-AKR/Gross virus-specific cytotoxic T lymphocytes. We report here the isolation and characterization of a replication-competent emv clone, pAK7, derived from an AKXL-5 mouse. This clone is novel in that it encodes a variant ecotropic murine leukemia virus that, when expressed in SC.Kb target cells, fails to be recognized efficiently by anti-AKR/Gross virus cytotoxic T lymphocytes. The pAK7 clone can therefore be used to further probe mechanisms of escape from cell-mediated immunity.

Animals↗
PubMed1999

In vitro induction of human immunodeficiency virus type 1 variants resistant to phosphoralaninate prodrugs of Z-methylenecyclopropane nucleoside analogues.

Two methylenecyclopropane nucleoside analogues with a phenylphosphoralaninate moiety, QYL-685 and QYL-609, exert potent and specific activities against human immunodeficiency virus type 1 strain LAI (HIV-1(LAI)) and HIV-2 in vitro. In this study, we induced HIV-1 variants resistant to QYL-685 by exposing HIV-1(LAI) to increasing concentrations of QYL-685. After 16 passages, the virus (HIV-1(P16)) was less sensitive to QYL-685 (104-fold), QYL-609 (>41-fold), and (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) (>1, 100-fold) than was HIV-1(LAI) and contained an M184I mutation. Two infectious clones, HIV-1(M184I) and HIV-1(M184V), were resistant to QYL-685, QYL-609, and 3TC, confirming that the M184I mutation was responsible for the observed resistance. Viral-fitness analyses (competitive HIV-1 replication assays) revealed that in the absence of drugs, M184I and M184V conferred a replication disadvantage on the virus compared to the replication efficiency of the wild-type infectious clone (HIV-1(wt)). However, in the presence of QYL-685 (4 microM), HIV-1(M184I) and HIV-1(M184V) showed greater fitness than HIV-1(wt). These data may provide structural and virological relevance with regard to the emergence of M184I and M184V substitutions in HIV-1.

2-Aminopurine↗
PubMed1983

Cloning of infectious adeno-associated virus genomes in bacterial plasmids.

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.

Adenoviruses, Human↗
PubMed1980

Infectious and noninfectious recombinant clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA.

Ten clones of Charon 4A containing proviruses of spleen necrosis virus, an avian retrovirus, and flanking chicken DNA sequences were isolated and characterized. Some clones gave rise to progeny with viral DNA sequences deleted or duplicated, probably as a result of crossing-over in the 600 bp terminal redundancy in viral DNA. The cellular sequences are different in each clone, indicating that all the proviruses are integrated in different sites in cellular DNA. Six clones are infectious and four are not. All the infectious molecules containing a provirus are of a similar size and are smaller than the noninfectious molecules containing a provirus. The viral DNA is not apparently different in eight clones, but two clones, one infectious and one noninfectious, lack two restriction sites each. Large changes in proviral DNA therefore do not seem responsible for the lack of infectivity of some clones. These results are consistent with the hypothesis that neighboring cellular DNA sequences control proviral expression (infectivity).

Animals↗
PubMed2004

Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2.

BACKGROUND: A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Delta30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. METHODS: A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Delta30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. RESULTS: The rDEN2Delta30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Delta30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Delta30, designated rDEN2Delta30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Delta30 in SCID-HuH-7 mice. CONCLUSIONS: The rDEN2Delta30 and rDEN2Delta30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

Aedes↗
PubMed1992

The establishment of rodent cell lines persistently producing HIV-1.

Animal cells differ in susceptibility to HIV-1 infection. To identify rodent cells which are permissive to HIV-1 replication, we transfected murine and rat cells with an infectious clone of HIV-1 and a vector containing the chloramphenicol acetyl transferase gene under the control of HIV-1 LTR. Three groups of transfectants were distinguished: (i) Cells which permit neither HIV-1 LTR activation nor viral protein expression; (ii) Cells which permit activation of the HIV-1 LTR but not HIV-1 protein expression; and (iii) Cells which are fully permissive to both HIV-1 LTR activation and virus production. The latter included rat embryonal fibroblastoid (Rat2) cells, which, in short-term transfection assays, produced titers of HIV-1 proteins similar to transfected T lymphoid cells. To establish persistently infected cells, Rat2 cells were stably transfected with a plasmid containing an infectious clone of HIV-1/N1T-A and a neo gene, yielding several G-418-resistant, HIV-1-producing cell cultures. Of these, Rat2/A1 and Rat2/A2 cell cultures expressed up to 60 ng HIV-1 p24 core antigen per 1 x 10(6) cells 3 days after cell subculture over a period of 3 months. Southern blot hybridization revealed that Rat2/A1 and Rat2/A2 carried one to two HIV-1 DNA copies per cell; no rearrangements or deletions in viral DNA were present. Restriction endonuclease analysis of HIV-1 DNA in Rat2/A2 cells suggested clonal expansion of cells containing integrated HIV-1 genome. Virus produced by the Rat2/A1 cells was infectious in human T cells. These data demonstrate that some rodent cells have no inherent restriction to persistent and efficient production of infectious HIV-1.

Animals↗
PubMed2003

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication.

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by approximately 100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an approximately 5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

Animals↗
PubMed2003

American genotype structures decrease dengue virus output from human monocytes and dendritic cells.

The dengue virus type 2 structures probably involved in human virulence were previously defined by sequencing the complete genome of both American and Southeast (SE) Asian genotype templates in patient serum (K. C. Leitmeyer et al., J. Virol. 73:4738-4747, 1999). We have now evaluated the effects of introducing a mutation in the envelope glycoprotein (E) gene and/or replacement of 5'- and 3'-nontranslated regions on dengue virus replication in human primary cell cultures. A series of chimeric infectious clones were generated containing different combinations of American and SE Asian genotype sequences. Some of the chimeric viruses had altered plaque morphology in mammalian cells; however, they replicated at similar rates in mosquito cells as measured by quantitative reverse transcription-PCR and plaque assay. Although susceptibility to virus infection varied from donor to donor in experiments using human macrophage and dendritic cells, we were able to measure consistent differences in viral RNA output per infected cell. Using this measurement, we demonstrated that the chimeric virus containing the E mutation had a lower virus output compared to the parental infectious clone. A larger reduction in virus output was observed for the triple mutant and the wild-type, American genotype virus from which chimeric inserts were derived. It appears that the three changes function synergistically, although the E mutation alone gives a lower output compared to the 5'- and 3'-terminal mutations. The data suggest that these changes may be responsible for decreased dengue virus replication in human target cells and for virulence characteristics during infection.

Animals↗
PubMed1991

Parvoviral target cell specificity: acquisition of fibrotropism by a mutant of the lymphotropic strain of minute virus of mice involves multiple amino acid substitutions within the capsid.

Unlike the prototype strain of minute virus of mice, MVM(p), the lymphotropic strain, MVM(i), cannot form plaques on monolayers of mouse A9 fibroblasts. At very low frequency, mutants arise in MVM(i) stocks which are able to plaque on A9 cells, and we report here the isolation and mapping of such a mutant, designated hr101. Analysis of intratypic recombinants containing regions of the hr101 genome substituted into the infectious clone of its parent MVM(i) shows that the ability to form plaques on fibroblast monolayers maps to the same small region of the coat protein gene which we had previously shown, by constructing intertypic recombinants, to contain the fibrotropic determinant of MVM(p) (Gardiner and Tattersall, J. Virol. 62, 2605-2613, 1988). DNA sequencing of the hr101 regions in virus stocks derived from these recombinants identified four single-base changes between the mutant coat protein gene and that of its parent. Each of these changes occurs in the same position as a similar change found between MVM(i) and MVM(p), and each of them change the amino acid encoded at that position. Three of the four changes substitute the same amino acid as found in MVM(p), and the fourth change substitutes an alanine in hr101 for a glutamic acid residue in MVM(i), in a position occupied by glycine in MVM(p). Analysis of the recombinants within this region shows that plaque formation on A9 monolayers is dependent upon the latter change plus one adjacent, MVM(p)-like change. This observation was confirmed by recreating this double mutant in the infectious clone of MVM(i) via site-directed mutagenesis. In addition to extending the host range of MVM(i) into A9 fibroblasts, the hr101 mutations have a complex effect on the virus' ability to grow lytically in a series of different T-lymphocyte cell lines.

Amino Acid Sequence↗
PubMed1995

Synthesis of a full-length infectious cDNA clone of cucurbit aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from other luteoviruses.

A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as an in vitro transcript, under control of a bacteriophage T7 RNA polymerase promoter, or in vivo, under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of its in vitro transcript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants cold be transmitted by the aphid vectors Myzus persicae and Aphis gossypii. The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.

Animals↗