Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene.
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PMID:Generation and characterization of the human immunodeficiency virus type 1 mutants. 170 90

The complete pol region of the simian immunodeficiency virus from African green monkeys was cloned and expressed in E. coli. The reverse transcriptase was purified to high specific activity and could be shown to contain both reverse transcriptase activity as well as an associated RNase H activity. As is observed with other reverse transcriptases the enzyme is composed of two subunits which cannot be separated by conventional techniques. When comparing the recombinant enzyme with the authentic enzyme isolated from virus no differences were found by biochemical, enzymological, or immunological criteria. Moreover, the action of inhibitors against this enzyme did not show significant differences when compared to reverse transcriptases from HIV-1 and HIV-2.
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PMID:Cloning and expression of the complete SIVagm pol region in E. coli. Purification and partial characterization of the reverse transcriptase. 170 22

Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27-pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity.
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PMID:Identification and characterization of human immunodeficiency virus type 1 gag-pol fusion protein in transfected mammalian cells. 170 86

Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity.
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PMID:Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products. 171 Dec 3

A 1.67-kb segment of the equine infectious anemia virus pol gene, encoding a 66-kDa reverse transcriptase (RT), was cloned and expressed in Escherichia coli. Recombinant RT, purified by a combination of metal chelate affinity chromatography and ion-exchange chromatography, displays both RNA-dependent DNA polymerase and RNase H activity. The affinity of purified RT for its replication primer, tRNA(3Lys) was equivalent to that observed for human immunodeficiency virus RT. Our data suggest that an additional domain between RT-RNase H and integrase on the equine infectious anemia virus pol open reading frame is not an integral component of the RT polypeptide.
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PMID:Purification and characterization of recombinant equine infectious anemia virus reverse transcriptase. 171 38

With the aid of monoclonal antibodies to the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1), low-molecular-mass subunits (p29, p32, and p40) were identified in HIV-1 RT purified from HIV (HTLV-IIIB) virions by isoelectric focusing. Epitope mapping with synthetic polypeptides from various regions of the pol gene suggests that the low-molecular-mass subunits result from N-terminal cleavage of the p51 subunit. The subunits could be separated only by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting. They could not be separated on chromatographic columns, suggesting that the subunits are complexed or conformationally arranged in such a way that their separation on the basis of molecular mass is not possible. The molecular mass of the active enzyme eluted from a chromatographic column (Sephacryl S-300) loaded with a mixture of the subunits was estimated to be 100 kDa.
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PMID:Epitope mapping of the low-molecular-mass subunits of reverse transcriptase in human immunodeficiency virus type 1 by monoclonal antibodies. 172 5

Drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) have been isolated by in vitro selection. MT-4 cells were infected with either a laboratory strain (HIV-IIIB) or a clinical isolate (no. 187) of HIV-1 and maintained in medium containing subeffective concentrations of the drugs 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI). By gradually increasing the drug concentration in the culture medium during propagation of the virus on fresh MT-4 cells, we were able to isolate variants of HIV-IIIB and clinical isolate 187 which showed up to 100-fold increases in resistance to the drugs. The drug resistance phenotypes remained stable after propagation of the variants in the absence of drug pressure for over 2 months. However, variants resistant to one drug showed little or no cross-resistance to the other, suggesting that the genetic bases for resistance to the compounds differed. Genotypic analysis of these nucleoside-resistant variants by polymerase chain reaction (PCR) with primer pairs previously shown to correspond to mutations responsible for resistance to AZT was also carried out. A heterogeneity of genotypes was observed, with known mutations at pol codons 70 and 215 occurring in most of the AZT-resistant variants generated from either HIV-IIIB or clinical strain 187. However, mutations in codons 67 and 219 were less frequently detected, and none of these changes were observed in each of four variants resistant to ddI. Cloning and sequencing studies of the reverse transcriptase coding region of two of the isolates were also performed and confirmed the PCR data that had been obtained. In addition to previously described mutation sites responsible for resistance to AZT, an HIV-IIIB-resistant variant was shown to be mutated at positions 108 (Val----Ala) and 135 (Ile----Thr), while a resistant variant of strain 187 was mutated at positions 50 (Ile----Val) and 135 (Ile----Val).
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PMID:In vitro selection of variants of human immunodeficiency virus type 1 resistant to 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine. 172 74

Deletion mutants of simian immunodeficiency virus (SIVmac) which were unable to integrate into host cells were generated by removing a portion of the integrase (IN) domain of the pol gene. The resulting plasmid was transfected into HUT-78 and human rhabdomyosarcoma cells. In comparison with the parental plasmid DNA transfected in parallel, the deletion mutant was found to direct efficient production of virus in both cell systems. Viruses derived from wild-type and mutant proviral DNAs were also tested for their relative replicative abilities in HUT-78 and U937 cells, and the kinetics of virus production was found to vary between these two cell systems. Analysis of DNA from infected cell nuclei showed that the deletion mutant lacked the ability to integrate despite being able to produce infectious virus. Using the sensitive polymerase chain reaction technique, we have clearly demonstrated the absence of the IN domain in the deletion mutant after infection and replication in HUT-78 cells. Such mutants might form the basis for the development of an experimental live attenuated vaccine.
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PMID:Generation of deletion mutants of simian immunodeficiency virus incapable of proviral integration. 841 98

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.
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PMID:Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells. 172 1

Under conditions in which a clonal cell line (M10) isolated from a human T cell lymphotrophic virus type I-transformed MT-4 cell line was completely killed by infection with wild-type human immunodeficiency virus type 1 (HIV-1), equivalent M10 cells survived infection with HIV-1 vif, vpr or vpu mutant virus after transient cytopathic effects. Several cell clones, which were isolated from the proliferating M10 cells after infection with vif and vpu mutant viruses (M10/vif- and M10/vpu-), had heterogeneous HIV-1 phenotypes in terms of HIV-1 antigen expression, their syncytium forming capacity, reverse transcriptase activity and the infectivity of HIV-1 particles produced. When the replication kinetics of the HIV-1 particles produced were assayed in M10 cells, the clones could be classified into three types, i.e. type I producing non-infectious HIV-1, type II producing infectious HIV-1 with low replicative ability and type III producing infectious HIV-1 with a replicative ability similar to that of wild-type HIV-1. HIV-1 major viral cell proteins and virus particle fractions were almost typical in types II and III but not in type I. Electron microscopic examination of particles released by I, II and III clones revealed rare defective, predominantly defective and essentially normal virions, respectively. Northern and Southern blot analyses revealed no apparent deletion in the proviral DNA and mRNA prepared from these clones, except in the case of type I and II clones isolated from M10/vpu- which contained large deletions in the mRNAs for gag and gag-pol proteins. Thus, M10 cells surviving infection with HIV-1 vif or vpu mutants are heterogeneous, persistently expressing HIV-1 antigens and producing non-infectious or less cytopathic virus.
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PMID:Cells surviving infection by human immunodeficiency virus type 1: vif or vpu mutants produce non-infectious or markedly less cytopathic viruses. 173 Sep 43


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