Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have studied the presence and significance of retroviral genome-derived DNA in the core of human immunodeficiency virus (HIV) particles produced from transfections of HXB2 expression vectors in COS-7 cells and from HIV type 1 IIIB chronically infected H9 cells. Viruses purified by sucrose cushion centrifugation and treated with DNase I contained 1000-fold more viral RNA than DNA. However, protease-defective viruses that contained only p160gag-pol had less than 100 times the amount of DNA in their cores than wild-type viruses suggesting that the p66/p51 form of reverse transcriptase was responsible for DNA transcription. Viruses produced by transfections in the presence of 3'-azido-3'-deoxythymidine (AZT) contained the viral RNA genome but only DNA of premature length because of the chain terminating effects of AZT. However such viruses were as infections for CD4+ cells as wild-type virus. We conclude that retrovirus-derived DNA in HIV-1 particles is not required for infection and does not play a significant role in this process.
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PMID:DNA found in human immunodeficiency virus type 1 particles may not be required for infectivity. 751 31

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).
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PMID:Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication. 752 16

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.
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PMID:The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein. 759 22

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.
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PMID:Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120. 760 10

By reverse transcriptase polymerase chain reaction on messenger RNA from human polymorphonuclear cells, we have isolated a sequence identical to the cDNA coding for intracellular interleukin 1 receptor antagonist (icIL-1ra), but containing an additional in-frame 63-bp sequence located three codons downstream of the translation start of icIL-1ra. This additional sequence is inserted between the first and second exon of the intracellular form, the latter of which is colinear with part of the first exon of the secreted form of IL-1ra. The additional sequence is coded by an extra exon located 2 kb downstream the first icIL-1ra-specific exon. The complementary DNA sequence of the alternatively spliced form of icIL-1ra shows that the predicted protein differs from classical icIL-1ra in the NH2 terminus by insertion of a leaderless sequence of 21 amino acids rich in glycine and glutamic acid residues. Transcripts coding for this new form of icIL-1ra were detected in activated fibroblasts, keratinocytes, and at low levels in myelomonocytic cells. The recombinant protein expressed in COS cells had an apparent molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis of 25 kD compared to 22 kD of classical icIL-1ra, and was mostly intracellular. The ability of this new form of icIL-1ra to inhibit IL-1 activity, in terms of induction of E-selectin and human immunodeficiency virus replication, was comparable to that of classical icIL-1ra. We propose to refer to this new form of icIL-1ra as icIL-1ra type II.
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PMID:Cloning and characterization of a new isoform of the interleukin 1 receptor antagonist. 762 20

The 96-amino acid Vpr protein is the only virion-associated regulatory protein encoded by the human immunodeficiency virus type 1 (HIV-1). Vpr incorporation into the viral particle is most likely due to an interaction with a viral structural protein. Recent data have shown that DNA encoding for the p55 Gag precursor protein (Pr55gag) is the minimal viral genetic information necessary for Vpr incorporation. Other studies have suggested that the p6 portion of Pr55gag, which is unique to lentiviruses, is involved in Vpr incorporation. To investigate the mechanism of incorporation of Vpr into HIV-1 virions, COS-7 cells were cotransfected with ptrENV, an expression vector that encodes all of the HIV-1 regulatory proteins including Rev and Vpr, and different constructs of pIIIgagCAR, a rev-dependent Gag expression plasmid that encodes Pr55gag and the viral protease. Virions produced from gag constructs containing a premature p6 termination codon at positions Leu-1, Ser-17, Tyr-36, or Leu-44 lacked detectable Vpr. In contrast, gag constructs with double Pro-10-Pro-11 substitutions for Leu-10-Leu-11 or a premature termination codon at position Pro-49 of p6 were still able to incorporate Vpr, however, with lower efficiency than wild type. The mutations described in this study affected directly two short regions within the p6 domain, which are highly conserved among primate immunodeficiency viruses. Our results suggest that the conserved (P-T/S-A-P-P) and (L-X-S-L-F-G) motifs located near the N-terminus and C-terminus, respectively, of the p6 domain of Gag are critical for Vpr incorporation into HIV-1 virions.
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PMID:Incorporation of Vpr into human immunodeficiency virus type 1: role of conserved regions within the P6 domain of Pr55gag. 764 78

Positive and negative effects of DNA replication on gene transcription have been documented in a variety of systems. We examined the effects of the simian virus 40 (SV40) origin of replication on transcription from the human immunodeficiency virus type 1 (HIV-1) promoter, using a transient expression assay in COS-1 cells. The basal activity and Tat transactivation of the HIV promoter were greatly stimulated by the SV40 origin of replication independent of its position relative to the long terminal repeat. These effects were abolished by mutational inactivation of the SV40 origin and were reduced by a DNA replication inhibitor. The magnitude of promoter activation exceeded the increment expected from the increase in template number resulting from DNA replication. The SV40 T-antigen-induced DNA replication augmented the generation of both processive and nonprocessive HIV long terminal repeat-directed transcripts, and Tat primarily enhanced the initiation of those transcripts that were destined to be efficiently elongated. Our data suggest that the HIV promoter displays greater transcriptional activity on replicative DNA templates. This property may influence the activity of integrated HIV provirus and its transition from latency to productive infection.
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PMID:Effects of the simian virus 40 origin of replication on transcription from the human immunodeficiency virus type 1 promoter. 781 9

A stable cell line encoding the sequences of all the human immunodeficiency virus type 1 proteins, with the exception of the gp160 envelope glycoprotein, was derived from transfection of monkey COS-7 cells. This cell line, referred to as CH-1, produces active viral protease that correctly processes its natural substrates and yields capsid particles. These particles contain reverse transcriptase activity and packaged viral RNA but are noninfectious. The level of expression of viral proteins is not toxic to the cells, yet it is comparable to that observed for chronically infected lymphocytes. These constitutively synthesized viral proteins provide a consistent system for the analysis of potential inhibitors of late viral functions. The lack of gp160 increases the biosafety of this assay system, while it allows the measurement of the effects on the production and release of capsid particles. A human immunodeficiency virus type 1 protease inhibitor was used to confirm the viral polyprotein maturation pathway in this system. Particles from cells treated with this protease inhibitor contain unprocessed p55gag precursor and have the same density as the mature particles. These immature particles contain viral RNA, but reverse transcriptase activity is significantly reduced. This cell line may serve to identify compounds that are able to affect viral assembly and maturation as well as to identify the interactions between the viral and cellular proteins involved in these essential processes.
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PMID:Constitutive production of nonenveloped human immunodeficiency virus type 1 particles by a mammalian cell line and effects of a protease inhibitor on particle maturation. 784 May 83

Previous studies have demonstrated that oligodeoxynucleotide phosphorothioates complementary to human immunodeficiency virus type 1 (HIV-1) RNA are more nuclease resistant and are effective inhibitors of HIV-1 replication than their unmodified counterpart. In this study, antisense oligodeoxynucleotide sequences were evaluated for therapeutic potential in the treatment of HIV infections. The use of HIV-infected lymphocytes to test the efficacy of a drug is very complex, and therefore it is difficult to draw conclusions about the mechanism. We used a COS-like Monkey kidney cell line (CMT3) stably transfected with plasmids pCMVgagpol-rre-r (containing gag and pol genes) and pCMVrev (containing the rev gene of HIV-1), derived from cDNA clone BH10, as a model. A biologically active provirus that transcribes and translates their nucleotide sequences into viral proteins p24, p39/41, p55, and p160 was generated. Sequence-specific and dose-dependent inhibition of HIV-1 viral protein synthesis and significant inhibition at the mRNA level were demonstrated by antisense construct GPI2A, directed against a nonregulatory region of the HIV-1 genome. Also, our studies demonstrated enhancement of the antisense effect through encapsulation in a cationic lipid preparation. The observed attenuation of HIV-1 mRNA levels suggests that, at least in part, the mechanism of action of GPI2A was at the transcript level. Further studies have also shown antiviral activity of this construct as determined by the reverse transcriptase assay using acutely and chronically infected cells of lymphoid origin (H9 cells). Toxicological studies involving cell growth characteristics, colony-forming ability, effects on cellular proteins, specific activities of labeled proteins, and DNA synthesis in cell culture showed no cytotoxic effects of GPI2A.
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PMID:Sequence-specific inhibition of gene expression by a novel antisense oligodeoxynucleotide phosphorothioate directed against a nonregulatory region of the human immunodeficiency virus type 1 genome. 785 19

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev, which is required for the cytoplasmic expression of unspliced and incompletely spliced viral mRNAs, is located predominantly in the nucleolus. In this study, we show that Rev translocates from the nucleolus to the cytoplasm in HeLa and COS cells transfected with Rev under conditions where rRNA synthesis is inhibited (e.g., with actinomycin D). Dominant-negative mutants with mutations in the activation domain of Rev, which are known to inhibit wild-type Rev function in trans, are unable to leave the nucleus upon actinomycin D treatment. More importantly, when present in excess, these mutants inhibit the translocation of wild-type Rev. This correlation of inhibitory activities suggests that Rev function depends on its transport to and presence (at least transient) in the cytoplasm. In this context, we discuss the possibility that Rev is actively involved in the transport of HIV-1-specific mRNAs containing the Rev response element (a highly structured RNA sequence, which is specifically recognized by the Rev trans-activator). We also discuss the potential of nucleocytoplasmic export of Rev as a target for anti-HIV chemotherapy.
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PMID:Nucleocytoplasmic transport of the Rev protein of human immunodeficiency virus type 1 is dependent on the activation domain of the protein. 786 18


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