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PubMed2003

[cDNAs cloning of SARS-CoV PUMC2 viral genome].

OBJECTIVE: To get the cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain. METHODS: Using the SARS-CoV PUMC2 strain genomic RNA as the template, the cDNA fragments were amplified by RT-PCR, the PCR products were further purified and ligated into the pGEM-T vector, and all the clones obtained were sequenced. RESULTS: The cDNA clones which cover the whole genome of SARS-CoV PUMC2 strain were obtained. CONCLUSIONS: These cDNAs can be provided for the function study of SARS-CoV proteins and the construction of full-length infectious cDNA clone of SARS-CoV.

Amino Acid Sequence↗
PubMed1989

T-lymphocyte clones responsive to Shigella flexneri.

T lymphocytes from a patient with Shigella flexneri dysentery and postdysenteric reactive arthritis were cloned by limiting dilution with recombinant interleukin-2 and a strain of S. flexneri different from that which had infected her. Five of eight clones produced proliferated in response to the shigellae used to generate the clones. The response required irradiated syngeneic blood mononuclear cells as antigen-presenting cells. One such clone, MC12, proliferated in response to both the shigellae used to generate the clones and the infecting shigellae but not to other shigellae, Salmonella heidelberg, or control Escherichia coli. MC12 was CD3+, CD4+, CD8-, and human histocompatibility leukocyte antigen (HLA)-DR+. The proliferative response to the shigellae was blocked by antibody to HLA-DR but not by antibody to HLA-A,B,C. The response required antigen-presenting cells that shared HLA-DR antigens with the clone and appeared to be restricted by HLA-DR2. The epitope recognized by MC12 was associated with the bacterial membranes. Thus, T-lymphocyte clones that proliferate in response to some shigellae can be isolated from patients with shigellosis.

Arthritis, Infectious↗
PubMed1989

Response of synovial fluid T cell clones to Yersinia enterocolitica antigens in patients with reactive Yersinia arthritis.

From synovial fluids of two patients with reactive arthritis following Yersinia enterocolitica infection, T lymphocyte clones were obtained that showed proliferative responses to Y. enterocolitica. The responses required autologous T-cell-depleted peripheral blood mononuclear cells as antigen presenting cells. Three clones were studied in detail; two of them showed a marked and specific response to Yersinia antigens alone, the other one recognized both Yersinia and Salmonella typhimurium antigens. The antigen-specific proliferation of the clones could be completely blocked by monoclonal antibodies to HLA-DR. These experiments show that synovial T lymphocytes presumably involved in the in situ immune response to microbial antigens triggering reactive arthritis can be cloned directly from the site of inflammation.

Adult↗
PubMed1992

Molecular characterization and heterogeneity of feline immunodeficiency virus isolates.

We have molecularly cloned the complete genomic DNA of TM2 strain of feline immunodeficiency virus (FIV) isolated in Japan and compared its nucleotide and the deduced amino acid sequence with those of previously described U.S. isolates, FIV Petaluma and FIV PPR. The infectious molecular clone of FIV TM2 is different from FIV Petaluma in host cell range; the clone can not infect Crandell feline kidney cells which were permissive for FIV Petaluma. The amino acid sequence homologies, in gag, pol, and env genes between FIV TM2 and Petaluma were 90%, 87%, and 81%, respectively. On the other hand, comparative analysis of each gene between FIV Petaluma and PPR showed 96,95, and 85%, respectively. These results suggested that the genomic diversity was present among FIV strains isolated from geographically distant areas. Interestingly, tat- and rev-like short open reading frames contained inframe stop codons in the FIV Petaluma but not in the FIV TM2.

Amino Acid Sequence↗
PubMed2000

Genome structure of Sagiyama virus and its relatedness to other alphaviruses.

Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3' poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3' terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.

Aedes↗
PubMed2004

A single amino acid change in gp41 is linked to the macrophage-only replication phenotype of a molecular clone of simian immunodeficiency virus derived from the brain of a macaque with neuropathogenic infection.

Simian immunodeficiency virus (SIV)-related neuropathogenesis has been observed in 90% of pig-tailed macaques infected with strain SIVsmmFGb, making it an excellent system for studying human immunodeficiency virus (HIV)-associated neurological disease. To investigate the genetics of SIV neurovirulence, infectious molecular clones were generated from the brain of a SIVsmmFGb-infected pig-tailed macaque. One clone, BPZm.12, displayed a macrophage-restricted phenotype not previously described; this clone replicated to high levels in macrophages, but did not replicate in peripheral blood mononuclear cells (PBMC) until at least 21 days postinfection. Sequence analysis of the env gene of BPZm.12 revealed the substitution of a serine residue for a highly conserved proline residue at position 629 in gp41. A mutant clone, which contained the conserved proline to serine (BPZm.12-629P), was able to replicate in both macrophages and PBMC without delay. A mutant of an unrelated dual tropic molecular clone PBj6.6, substituting proline for serine (PBj6.6-629S), replicated to high levels in macrophages, but did not replicate in PBMC at any time point. These data indicated that a single determinant in gp41 of an SIV clone changed its phenotype from macrophage tropic to dual tropic.

Amino Acid Sequence↗
PubMed2006

HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry.

Human immunodeficiency virus type 1 (HIV-1) infection can be inhibited by small molecules that target the CCR5 coreceptor. Here, we describe some properties of clonal viruses resistant to one such inhibitor, SCH-D, using both chimeric, infectious molecular clones and Env-pseudotypes. Studies using combinations of CCR5 ligands, including small molecule inhibitors, monoclonal antibodies (MAbs) and chemokine derivatives such as PSC-RANTES, show that the fully SCH-D-resistant viruses enter target cells by using the SCH-D-bound form of CCR5. However, the way resistance to SCH-D and other small molecule CCR5 inhibitors is manifested depends on the target cell and the nature of the assay (single- vs. multi-cycle). In multi-cycle assays using primary lymphocytes, SCH-D does not inhibit resistant molecular clones, and it can even enhance their infectivity modestly. In contrast, the same viruses (as Env-pseudotypes) are significantly inhibited by SCH-D in single-cycle entry assays using U87-CD4/CCR5 cells, resistance being manifested by incomplete inhibition at high SCH-D concentrations. When a single-cycle, Env-pseudotype entry assay was performed using either U87-CD4/CCR5 cells or PBMC under comparable conditions, entry was inhibited by up to 88% in the former cells but by only 28% in the PBMC. Hence, there are both cell- and assay-dependent influences on how resistance is manifested. We also take this opportunity to correct our previous report that SCH-D-resistant isolates are also substantially cross-resistant to PSC-RANTES [Marozsan, A.J., Kuhmann, S.E., Morgan, T., Herrera, C., Rivera-Troche, E., Xu, S., Baroudy, B.M., Strizki, J., Moore, J.P., 2005. Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D). Virology 338 (1), 182-199]. A substantial element of this resistance was attributable to the unappreciated carry-over of SCH-D from the selection cultures into analytical assays.

CCR5 Receptor Antagonists↗
PubMed1995

RNA transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require VpG for infectivity.

Feline calicivirus (FCV) is a positive-strand, nonenveloped RNA virus in the family Caliciviridae. A cDNA library of the Urbana (URB) strain of FCV was generated and the sequence of the genome was determined from overlapping clones except for 13 bases from the 5'-end. The 5'-end sequence was identified by analysis of clones derived by RT-PCR across the ligated 5'- and 3'-ends of the RNA genome. A full-length cDNA clone of the RNA genome of the URB strain was constructed and placed downstream of the T7 RNA polymerase promoter and RNA transcripts generated in vitro from this clone were infectious when introduced into feline kidney cells. A virus-encoded genome-linked protein, VpG, which is considered to be essential for infectivity of wild-type genomic FCV RNA, was not required for the initiation of FCV infection by the synthetic transcripts. However, the addition of a cap structure analog (m7G(5')ppp(5')G) during in vitro transcription of the synthetic RNA was necessary for successful virus recovery. Two silent mutations engineered into the full-length clone were identified in the genomic RNA from recovered progeny virus. This system of introducing site-specific genetic changes into the genome of feline calicivirus and the recovery of infectious mutant viruses will enable studies related to the molecular basis for replication, growth restriction, and pathogenicity of this and other members of the Caliciviridae.

Animals↗
PubMed1989

Mutational analysis of the human immunodeficiency virus vpr open reading frame.

Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.

Cytopathogenic Effect, Viral↗
PubMed1993

Analysis of envelope changes acquired by SIVmac239 during neuroadaption in rhesus macaques.

Nucleotide sequence analyses of the env genes of two neurotropic variants of SIVmac239 were performed to determine whether molecular changes in these genes could be correlated with neurotropism. Biological characterization of virus from the infectious molecular clone of SIVmac239 had shown that it is highly lymphocyte-tropic and poorly macrophage-tropic. This virus failed to replicate in the brain after intracerebral inoculation, but passage of this virus in macaques resulted in development of viral variants that had acquired cell tropism for macrophages and were neurovirulent (D. P. Sharma, M. C. Zink, H. Anderson, R. J. Adams, J. E. Clements, S. V. Joag, and O. Narayan, J. Virol., 66, 3550-3556, 1992). The neurotropic virus SIVmac239/R71 was obtained from the brain of a monkey after the third in vivo passage of SIVmac239. Inoculation of this virus into another macaque leads to CNS disease and the isolation of another neurotropic virus SIVmac239/17E. The viral env sequences obtained by polymerase chain reaction amplification directly from DNA obtained from the brain of R71 and 17E macaques had a limited number of changes dispersed throughout the env gene when compared to the parental virus, SIVmac239. The most important finding was that there was a common set of nucleotide changes in the env gene of both R71 and 17E. This suggested that viruses containing these changes had a selective growth advantage in the brain and were the predominant species present in the central nervous system of macaques R71 and 17E. Analysis of individual clones containing the R71 env gene revealed that different env genes were present, but all had the changes that were conserved in both R71 and 17E but not present in the original lymphocyte-tropic parental virus, SIVmac239. Construction of an infectious recombinant virus containing the tat, rev, and env genes from 17E and the remainder of the genome from the parental virus SIVmac239 resulted in a virus that had the macrophage-tropism of 17E virus isolated from brain. This demonstrates that the env gene of 17E confers the cellular tropism of the virus on the parental virus, SIVmac239.

Amino Acid Sequence↗
PubMed2001

Coronavirus derived expression systems.

Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient (>20 microg ml(-1)) and stable (>20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy.

Coronavirus↗
PubMed1999

Molecular cloning and cell-specific growth characterization of polymorphic variants of type D serogroup 2 simian retroviruses.

Simian retroviruses (SRVs), the etiological agent of a spontaneous Simian acquired immunodeficiency syndrome, endemically infects large percentages of Asian macaques housed in biomedical research colonies and severely compromises the effective use of these species as a viable research animal. We recently described the molecular cloning of a serogroup 2 SRV, D2/RHE/OR, which causes mild immunosuppression in rhesus macaques. A restriction site variant, D2/RHE/OR/V1, has also been recovered from severely ill animals endemically infected with D2/RHE/OR. We now report the complete nucleotide sequences of D2/RHE/OR and D2/RHE/OR/V1. Both infectious molecular clones retain the genetic structure typical of type D SRVs (5' LTR-gag-prt-pol-env-3'LTR) and encode identically sized 8105-bp proviruses. D2/RHE/OR and D2/RHE/OR/V1 are 99.3% similar at the amino acid level, exhibiting only 17 residue differences, of which 10 are located in the envelope glycoproteins. The molecular clones and reciprocal chimeric viruses were used to assess the contribution of different genetic domains to virus infectivity in a T cell infection assay. These experiments indicate that D2/RHE/OR has a reduced ability to infect specific T cell lines, especially Hut-78 and MT-4 cells, and that the envelope gene is not the sole determinant of in vitro tropism.

Amino Acid Sequence↗
PubMedDate not supplied

A molecular clone of HTLV-III with biological activity.

Acquired immune deficiency syndrome (AIDS) is an epidemic immunosuppressive disease characteristically associated with a depletion of T lymphocytes of the helper/inducer phenotype. Numerous converging lines of research have implicated a human T-cell lymphotropic retrovirus, HTLV-III, in the pathogenesis of AIDS. Recently, several distinct forms of the HTLV-III genome were molecularly cloned in phage and extensively characterized. In the present study, a clone containing full-length HTLV-III proviral DNA was inserted into a plasmid and used to transfect cord blood T cells from normal newborn humans. We demonstrate that this molecular clone is infectious in vitro and causes marked cytopathic effects on T-cell cultures. This is the first direct evidence that the HTLV-III genome, rather than a minor component of the virus complex, is cytopathic for T cells. Using this biologically competent clone and mutants derived from it, it should now be possible to localize the subgenomic regions that contribute to the biological effects of HTLV-III.

Acquired Immunodeficiency Syndrome↗
PubMed2026

A newly established reverse genetic system for a circular RNA virus reveals new requirements for infection and its biocontrol potential.

Ambiviruses are fungal-infecting circular RNA viruses that uniquely combine viroid-like and viral features, yet the function of their conserved ORF-B protein and their effects on hosts remain unknown, hindered by the lack of a reverse genetics system. Here, we constructed the first infectious cDNA clone of an ambivirus, Fusarium graminearum ambivirus 1 (FgAV1), using a head-to-tail dimer placed downstream of a fungal promoter. FgAV1 was horizontally transmitted via hyphal anastomosis to virus-free Fusarium graminearum strains. Notably, a reverse-oriented dimer construct was also infectious and transmissible. Targeted mutagenesis revealed that both ORF-A- and ORF-B-encoded proteins and the presence of embedded ribozymes are indispensable for ambivirus replication. Our results further demonstrate that FgAV1 infection triggers a fungal RNAi response, extending the antiviral role of host sRNAs to circular RNA viruses. Furthermore, FgAV1 infection suppressed fungal growth and significantly reduced the virulence of F. graminearum on wheat. These findings provide novel insights into ambivirus replication and their potential in fungal pathogen biocontrol.

RNA Viruses↗
PubMed2001

Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees.

Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the approximately 20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts ( approximately 0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population.

Animals↗
PubMed1998

Cleavage of the feline calicivirus capsid precursor is mediated by a virus-encoded proteinase.

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3'-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1' position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.

Animals↗
PubMed2000

Analysis of HIV type 1 reverse transcriptase: comparing sequences of viral isolates with mutational data.

A result of the high level of mutagenesis during HIV-1 viral replication is that many, if not most, HIV-1 virions and proviruses are defective and are not infectious. There is a vast amount of HIV-1 sequence data available. Unless any particular sequence is shown to be from a stable DNA clone (e.g., lambda) that can transfect cells and produce virions, then it is not known if that sequence was from an infectious HIV-1. Most sequences have not been shown to be from infectious clones. We have reported a saturation mutagenesis of a 109-amino acid region of the HIV-1 reverse transcriptase, in which we assayed the effects of 366 single-amino acid substitutions. We examined a set of sequences in the Los Alamos HIV-1 sequence database. We found that none of the sequences derived from stable infectious clones had substitutions that produce an inactive reverse transcriptase. However, we found that other sequences in this database had substitutions that inactivate the reverse transcriptase. We predict that these sequences are not from infectious clones. This method may also be useful for evaluating the sequences of other viruses.

Amino Acid Sequence↗