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PubMed1988

Molecular evolution of the human and simian immunodeficiency viruses.

Molecular evolution and phylogeny of different human immunodeficiency virus type 1 (HIV1) strains, of a type 2 (HIV2) strain, and of two simian immunodeficiency viruses (SIVAGM and SIVMAC) have been studied by comparing the nucleotide sequences of the two regions of their pol genes which encode the reverse transcriptase (RT) and endonuclease/integrase (EN). The analyses show that the different HIV 1s form one cluster (HIV1 group) and that the SIVs and HIV2 form another (HIV2 group). When the entire genomes of a HIV1, a HIV2, and the two SIVs were compared, the SIVAGM showed a unique pattern of mutation accumulations; that is, the SIVAGM has accumulated more nonsynonymous changes than synonymous changes in the RT and EN regions after its recent divergence from SIVMAC-142, and, furthermore, it has a deletion of approximately 350 bp in the region between the pol and env genes. The SIVAGM was apparently derived from cell cultures infected with a macaque isolate, SIVMAC-251. The contamination provides an opportunity to measure the maximum rate of evolution in the SIVAGM by comparing its DNA sequence to those of SIVMAC-251 and SIVMAC-142. The analysis shows that the rates are given approximately by (1.95 +/- 1.37) x 10(-3)/site/year for one SIVAGM sequence and (5.18 +/- 2.25) x 10(-3)/site/year for another.

Base Sequence↗
PubMed1991

A peptide library expressed in yeast reveals new major epitopes from human immunodeficiency virus type 1.

In order to characterize novel human immunodeficiency virus type 1 (HIV-1) continuous epitopes, we designed a simple method, based on recombinant DNA, providing a complete set of peptides derived from HIV-1. A library (4 x 10(4) clones) was first constructed in a new expression/secretion vector, using as inserts small fragments of HIV-1 DNA (50-150 bp) generated by random DNAse I cleavage. This peptide library, expressed in the yeast Saccharomyces cerevisiae, was screened with sera of HIV-1 infected individuals and human and murine anti-HIV-1 monoclonal antibodies. Plasmids from immunoreactive colonies were recovered and the sequences of the HIV-1 derived inserts were determined. By using human sera, we have detected classical HIV-1 epitopes and identified two novel major epitopes, which may be used to improve diagnostic tests, localized in the p24 core protein and in the endonuclease. In addition, four minor epitopes were also defined by screening the library with monoclonal antibodies: in the protease, in the p17 core protein, in gp120 and near the C-terminal of gp41. This method is general and can be used for any protein from which a cloned cDNA is available.

Amino Acid Sequence↗
PubMed2001

Selective transcription and modulation of resting T cell activity by preintegrated HIV DNA.

The quiescent nature of most peripheral T cells poses an effective limitation to human immunodeficiency virus (HIV) replication and, in particular, to viral integration into the host chromatin. Two HIV proteins, Nef and Tat, increase T cell activity, but a requirement of integration for viral gene expression would preclude a role for these proteins in resting cells. Here, we report that HIV infection leads to selective transcription of the nef and tat genes before integration. This preintegration transcription in quiescent cells leads to increased T cell activation and viral replication.

Anti-HIV Agents↗
PubMed1991

Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.

Amino Acid Sequence↗
PubMed2002

Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection.

Most current evidence suggests that the infection of resting CD4(+) T cells by human immunodeficiency virus type 1 (HIV-1) is not productive due to partial or complete blocks in the viral life cycle at steps prior to integration of the viral genome into the host cell chromosome. However, stimulation of an infected resting T cell by antigen, cytokines, or microenvironmental factors can overcome these blocks and allow for the production of progeny virions. In this study, we sought to understand the structure and fate of the virus in unstimulated resting CD4(+) T cells. Using a novel linker-mediated PCR assay designed to detect and characterize linear unintegrated forms of the HIV-1 genome, we demonstrate that reverse transcription can proceed to completion following the infection of resting T cells, generating the substrate for the retroviral integration reaction. However, reverse transcription in resting T cells is far slower than in activated T cells, requiring 2 to 3 days to complete. The delay in completing reverse transcription may make the viral DNA genome more susceptible to competing decay processes. To explore the relationship between the formation of the linear viral genome and the stability of the preintegration state, we employed a recombinant HIV-1 virus expressing the enhanced green fluorescent protein to measure the rate at which HIV-1 decays in the preintegration state. Our results demonstrate that the preintegration state is labile and decays rapidly (half-life = 1 day) following the entry of HIV-1 into a resting T cell, with significant decay occurring during the slow process of reverse transcription.

Base Sequence↗
PubMed2004

Evidence for gene expression by unintegrated human immunodeficiency virus type 1 DNA species.

The integrated form of human immunodeficiency virus type 1 (HIV-1) DNA is classically considered to be the sole template for viral gene expression. However, several studies have suggested that unintegrated viral DNA species could also support transcription. To determine the contribution of the different species of HIV-1 DNA to viral expression, we first monitored intracellular levels of various HIV-1 DNA and RNA species in a single-round infection assay. We observed that, in comparison to the precocity of HIV-1 DNA synthesis, viral expression was delayed, suggesting that only the HIV-1 DNA species that persist for a sufficient period of time would be transcribed efficiently. We next evaluated the transcriptional activity of the circular forms of HIV-1 DNA bearing two long terminal repeats, since these episomes were reported to exhibit an intrinsic molecular stability. Our results support the notion that these circular species of HIV-1 DNA are naturally transcribed during HIV-1 infection, thereby participating in virus replication.

Cell Line↗
PubMed1997

Intracellular antibodies (intrabodies) for gene therapy of infectious diseases.

Intracellular antibodies (intrabodies) represent a new class of neutralizing molecules with a potential use in gene therapy. Intrabodies are engineered single-chain antibodies in which the variable domain of the heavy chain is joined to the variable domain of the light chain through a peptide linker, preserving the affinity of the parent antibody. Intrabodies are expressed inside cells and directed to different subcellular compartments where they can exert their function more effectively. The effects of intrabodies have been investigated using structural, regulatory, and enzymatic proteins of the human immunodeficiency virus (HIV-1) as targets. These intrabodies have demonstrated their versatility by controlling early as well as late events of the viral life cycle. In this article, we review studies of the use of intrabodies as research tools and therapeutic agents against HIV-1.

Antibody Formation↗
PubMed2006

HIV-1 inactivation by nucleic acid aptamers.

Although developments in small-molecule therapeutics for HIV-1 have been dramatic in recent years, the rapid selection of drug-resistant viral strains and the adverse side effects associated with long-term exposure to current treatments propel continued exploration of alternative anti-HIV-1 agents. Non-coding nucleic acids have emerged as potent inhibitors that dramatically suppress viral function both in vitro and in cell culture. In particular, RNA and DNA aptamers inhibit HIV-1 function by directly interfering with essential proteins at critical stages in the viral replication cycle (Figure 1). Their antiviral efficacy is expected to be a function, in part, of the biochemical properties of the aptamer-target interaction. Accordingly, we present an overview of biochemical and cell culture analyses of the expanding list of aptamers targeting HIV-1. Our discussion focuses on the inhibition of viral enzymes (reverse transcription, proteolytic processing, and chromosomal integration), viral expression (Rev/RRE and Tat/TAR), viral packaging (p55Gag, matrix and nucleocapsid), and viral entry (gp120) (Table 1). Additional nucleic acid-based strategies for inactivation of HIV-1 function (including RNAi, antisense, and ribozymes) have also demonstrated their utility. These approaches are reviewed in other chapters of this volume and elsewhere.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Molecular bases for the anti-HIV-1 effect of NO. Commentary.

In infected human cells, nitric oxide (NO) has been shown to inhibit the replication of the human immunodeficiency virus-1 (HIV-1), the etiological agent of AIDS. Evidence suggests that NO may regulate HIV-1 replication by affecting the sulphydryl redox state. In this respect, it has been very recently demonstrated that NO-donors inactivate the HIV-1-encoded protease and reverse transcriptase in vitro. Further viral and host NO targets may be envisaged. Although no data are available on the anti-HIV-1 effect of NO in vivo, NO-releasing drugs, clinically used in the treatment of cardiovascular disorders, may represent a novel class of molecules for decreasing virus replication. Here, the possible molecular bases for the anti-HIV-1 effect of NO are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Obstacles to successful antiretroviral treatment of HIV-1 infection: problems & perspectives.

Mutations in human immunodeficiency virus type 1 (HIV-1) are a major impediment to successful highly active antiretroviral therapy (HAART) and the design of anti-HIV vaccines. Although HAART has made long-term suppression of HIV a reality, drug resistance, drug toxicity, drug penetration, adherence to therapy, low levels of continued viral replication in cellular reservoirs and augmentation of host immune responses are some of the most important challenges that remain to be sorted out. Continuing viral replication in the face of HAART leads to the accumulation of drug resistance mutations, increase in viral loads and eventual disease progression. Patients who fail therapy have minimal options for their clinical management. Therefore, a clear understanding of the pathogenesis of drug-resistant HIV-1, and all of the issues that influence the success of HAART is urgently needed. In the present article, we discuss various obstacles to HIV therapy, and provide perspectives relating to these issues that are critical in determining the success or failure of HAART.

Anti-HIV Agents↗
PubMed2004

Potential drug resistance polymorphisms in the integrase gene of HIV type 1 subtype A.

Variation in HIV-1 genes within and between subtypes has been best defined in the env gene, however, other more conserved genes vary between subtypes. Integrase (IN) and other regions of the pol gene are highly conserved due to their integral role in HIV replication and therefore are targets for antiviral drugs. In this study 3 individuals, infected heterosexually with HIV-1 subtype A, were examined for IN polymorphisms. Two patients' sequences clustered phylogenetically with other subtype A sequences and one patient's sequence was most similar to the circulating recombinant form CRF_02. No polymorphisms were observed in either of the motifs containing residues critical residues for IN activity. Polymorphisms were observed in a residue associated with resistance to anti-integrase drugs. In addition, a number of unique polymorphisms were observed in one individual (WM1666). IN can vary significantly within a subtype as well as between subtypes, and mutations associated with resistance to anti-integrase compounds can be present in drug naive individuals.

Amino Acid Sequence↗
PubMed2006

Sensitive and specific LC-ESI-MS/MS method for the determination of a styrylquinoline, BA011FZ041, a potent HIV anti-integrase agent, in rat plasma.

A LC-MS/MS method was validated for the determination of BA011FZ041, a styrylquinoline derivative. After addition of BA011FZ055 as internal standard (IS), the method involved solid phase extraction (SPE), LC separation with an ether-phenyl column and quantification by MS/MS after positive ESI. The calibration curve, ranging from 1 to 500 ng/mL was fitted to a 1/x-weighted quadratic regression model. Lower limit of quantification (LLOQ) was 1 ng/mL using 100 microL of plasma. Intra- and inter-assay precision and accuracy values were within the regulatory limits. The method was successfully applied to the determination of BA011FZ041 in rat plasma and PBMCs after i.v. dosing.

Animals↗
PubMed2000

Retroviral integrase inhibitors year 2000: update and perspectives.

HIV-1 integrase is an essential enzyme for retroviral replication and a rational target for the design of anti-AIDS drugs. A number of inhibitors have been reported in the past 8 years. This review focuses on the recent developments in the past 2 years. There are now several inhibitors with known sites of actions and antiviral activity. The challenge is to convert these leads into drugs that will selectively target integrase in vivo, and can be added to our antiviral armamentarium.

Anti-HIV Agents↗
PubMed1999

A novel Vpr peptide interactor fused to integrase (IN) restores integration activity to IN-defective HIV-1 virions.

A novel approach to complement human immunodeficiency virus type I (HIV-1) integrase (IN)-defective virions has been identified. The approach involves fusion of a 23-amino-acid stretch to the N-terminus of wild-type IN and coexpression of this chimera with the IN-defective proviral template in virus producing cells. The 23-amino-acid peptide represents a Vpr "interactor," referred to as the the WxxF or WF domain, which apparently leads to docking of the domain along with the fusion partner onto HIV-1 Vpr, thus permitting virion incorporation of the chimeric protein when expressed, in trans, with other viral products. Transfection of the WF-IN expression plasmid along with HIV-1 viral clones that produce Vpr, but bear an IN mutation, results in the release of a proportion of viral particles that are competent for integration. The extent of complementation was assessed using the MAGI cell assay, where integration of viral DNA results in the eventual appearance of easily visible multinucleated blue syncytia. The efficiency of dWF-IN (double copy of WF domain) complementation is not improved markedly by incorporation of a HIV-1 protease cleavage site (PR) between the dWF domain and IN (dWF-PR-IN), unlike that observed with Vpr fusions to IN. Furthermore, the ability of Vpr-PR-IN and dWF-PR-IN to complement IN-defective proviral clones, both of which bear an intervening protease cleavage site, appear comparable. Western blotting analyses using virions isolated through sucrose cushions demonstrate clearly the incorporation of the dWF-IN fusion protein into Vpr containing HIV-1 particles but not in Vpr-deficient virions. Additional Western blotting analyses indicate that all Vpr-IN and dWF-IN chimeras, with or without a PR site, are packaged into virions. The efficiency of virion incorporation of Vpr-IN and dWF-IN chimeras appears approximately comparable by Western blotting analysis. The ability of dWF-IN to complement IN-defective proviruses with efficiency similar to that of Vpr-PR-IN and dWF-PR-IN indicates that dWF-IN retains the full complement of functions necessary for integration of proviral DNA and is likely due to the benign nature of this small domain at the amino-terminus of IN.

Artificial Gene Fusion↗
PubMed2006

Regiocontrolled synthesis and HIV inhibitory activity of unsymmetrical binaphthoquinone and trimeric naphthoquinone derivatives of conocurvone.

Unsymmetrical biquinone and trimeric quinone derivatives were synthesized using halotriflate-biselectrophilic naphthoquinones through stepwise regioselective quinone substitution chemistry and evaluated for their ability to inhibit the cytopathogenic effects of HIV-1 using an MTT colorimetric assay. Compounds were also screened for their ability to inhibit the activity of HIV-1 integrase in vitro. Pyranylated trimeric quinones and biquinones exhibited both antiviral activity and integrase inhibitory activity. Conocurvone 1 and trimeric quinone 21 were the most potent HIV integrase inhibitors in the series. All of the biquinones showed HIV inhibitory activity. Simple methoxy substituted biquinones did not inhibit HIV-1 integrase.

Anti-HIV Agents↗
PubMed2002

Inhibition of human immunodeficiency virus type 1 integration by diketo derivatives.

A series of diketo derivatives was found to inhibit human immunodeficiency virus type 1 (HIV-1) integrase activity. Only L-708,906 inhibited the replication of HIV-1(III(B)) (50% effective concentration, 12 micro M), HIV-1 clinical strains, HIV-1 strains resistant to reverse transcriptase or fusion inhibitors, HIV-2 (ROD strain) and simian immunodeficiency virus (MAC(251)). The combinations of L-708,906 with zidovudine, nevirapine, or nelfinavir proved to be subsynergistic. In cell culture, addition of L-708,906 could be postponed for 7 h after infection, a moment coinciding with HIV integration. Inhibition of integration in cell culture was confirmed by quantitative Alu-PCR.

Acetoacetates↗