scholarly journals Oxadiazols: a New Class of Rationally Designed Anti-Human Immunodeficiency Virus Compounds Targeting the Nuclear Localization Signal of the Viral Matrix Protein

2005 ◽  
Vol 79 (20) ◽  
pp. 13028-13036 ◽  
Author(s):  
Omar Haffar ◽  
Larisa Dubrovsky ◽  
Richard Lowe ◽  
Reem Berro ◽  
Fatah Kashanchi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Matrix Protein ◽  
Computer Assisted ◽  
Viral Dna ◽  
Immunodeficiency Virus ◽  
Localization Signal ◽  
Hiv 1

ABSTRACT Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.

scholarly journals Nuclear Import of the Preintegration Complex Is Blocked upon Infection by Human Immunodeficiency Virus Type 1 in Mouse Cells

2006 ◽  
Vol 81 (2) ◽  
pp. 677-688 ◽  
Author(s):  
Naomi Tsurutani ◽  
Jiro Yasuda ◽  
Naoki Yamamoto ◽  
Byung-Il Choi ◽  
Motohiko Kadoki ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Localization ◽  
Reverse Transcription ◽  
Nuclear Import ◽  
Viral Dna ◽  
Preintegration Complex ◽  
Immunodeficiency Virus ◽  
Mouse Cells ◽  
Hiv 1

ABSTRACT Mouse cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of host range barriers at steps including virus entry, transcription, RNA splicing, polyprotein processing, assembly, and release. The exact mechanisms for the suppression, however, are not completely understood. To elucidate further the barriers against HIV-1 replication in mouse cells, we analyzed the replication of the virus in lymphocytes from human CD4/CXCR4 transgenic mice. Although primary splenocytes and thymocytes allowed the entry and reverse transcription of HIV-1, the integration efficiency of the viral DNA was greatly reduced in these cells relative to human peripheral blood mononuclear cells, suggesting an additional block(s) before or at the point of host chromosome integration of the viral DNA. Preintegration processes were further analyzed using HIV-1 pseudotyped viruses. The reverse transcription step of HIV-1 pseudotyped with the envelope of murine leukemia virus or vesicular stomatitis virus glycoprotein was efficiently supported in both human and mouse cells, but nuclear import of the preintegration complex (PIC) of HIV-1 was blocked in mouse cells. We found that green fluorescent protein (GFP)-labeled HIV-1 integrase, which is known to be important in the nuclear localization of the PIC, could not be imported into the nucleus of mouse cells, in contrast to human cells. On the other hand, GFP-Vpr localized exclusively to the nuclei of both mouse and human cells. These observations suggest that, due to the dysfunction of integrase, the nuclear localization of PIC is suppressed in mouse cells.

scholarly journals Class II Integrase Mutants with Changes in Putative Nuclear Localization Signals Are Primarily Blocked at a Postnuclear Entry Step of Human Immunodeficiency Virus Type 1 Replication

2004 ◽  
Vol 78 (23) ◽  
pp. 12735-12746 ◽  
Author(s):  
Richard Lu ◽  
Ana Limón ◽  
Eric Devroe ◽  
Pamela A. Silver ◽  
Peter Cherepanov ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nuclear Localization ◽  
Reverse Transcription ◽  
Nuclear Import ◽  
Class Ii ◽  
Class I ◽  
Nuclear Localization Signals ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Integrase has been implicated in human immunodeficiency virus type 1 (HIV-1) nuclear import. Integrase analyses, however, can be complicated by the pleiotropic nature of mutations: whereas class I mutants are integration defective, class II mutants display additional assembly and/or reverse transcription defects. We previously determined that HIV-1V165A, originally reported as defective for nuclear import, was a class II mutant. Here we analyzed mutants containing changes in other putative nuclear localization signals, including 186KRK188/211KELQKQITK219 and Cys-130. Previous work established HIV-1K186Q, HIV-1Q214L/Q216L, and HIV-1C130G as replication defective, but phenotypic classification was unclear and nuclear import in nondividing cells was not addressed. Consistent with previous reports, most of the bipartite mutants studied here were replication defective. These mutants as well as HIV-1V165A synthesized reduced cDNA levels, but a normal fraction of mutant cDNA localized to dividing and nondividing cell nuclei. Somewhat surprisingly, recombinant class II mutant proteins were catalytically active, and class II Vpr-integrase fusion proteins efficiently complemented class I mutant virus. Since a class I Vpr-integrase mutant efficiently complemented class II mutant viruses under conditions in which class II Vpr-integrases failed to function, we conclude that classes I and II define two distinct complementation groups and suggest that class II mutants are primarily defective at a postnuclear entry step of HIV-1 replication. HIV-1C130G was also defective for reverse transcription, but Vpr-integraseC130G did not efficiently complement class I mutant HIV-1. Since HIV-1C130A grew like the wild type, we conclude that Cys-130 is not essential for replication and speculate that perturbation of integrase structure contributed to the pleiotropic HIV-1C130G phenotype.

scholarly journals Wild-Type Levels of Nuclear Localization and Human Immunodeficiency Virus Type 1 Replication in the Absence of the Central DNA Flap

2002 ◽  
Vol 76 (23) ◽  
pp. 12078-12086 ◽  
Author(s):  
Ana Limón ◽  
Noriko Nakajima ◽  
Richard Lu ◽  
Hina Z. Ghory ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Numerous factors have been implicated in the nuclear localization of retroviral preintegration complexes. Whereas sequences in human immunodeficiency virus type 1 (HIV-1) matrix, Vpr, and integrase proteins were initially reported to function specifically in nondividing cells, other recently identified sequences apparently function in dividing cells as well. One of these, the central DNA flap formed during reverse transcription, is specific to lentiviruses. It was previously reported that flap-negative (F−) HIV-1LAI was completely defective for viral spread in the MT-4 T-cell line, yet F− HIV-1 vectors were only 2- to 10-fold defective in various single-round transduction assays. To address these different findings, we analyzed the infectivity and nuclear localization phenotypes of two highly related T-cell-tropic strains, HIV-1NL4-3 and a derivative of HIV-1HXBc2 deficient for both Vpr and Nef. In stark contrast to the previous report, F− derivatives of both strains replicated efficiently in MT-4 cells. F− HIV-1NL4-3 also spread like wild-type HIV-1NL4-3 in infected Jurkat and primary T-cell cultures. In contrast, F− HIV-1HXBc2 was replication defective in primary T cells. Results of real-time quantitative PCR assays, however, indicated that F− HIV-1HXBc2 entered primary T-cell nuclei as efficiently as its wild-type counterpart. Thus, the F− HIV-1HXBc2 growth defect did not appear to correlate with defective nuclear import. Consistent with this observation, wild-type nef restored replication to F− HIV-1HXBc2 in primary T cells. Our results indicate that the central DNA flap does not play a major role in either preintegration complex nuclear import or HIV-1 replication in a variety of cell types.

scholarly journals Nuclear Localization of Human Immunodeficiency Virus Type 1 Preintegration Complexes (PICs): V165A and R166A Are Pleiotropic Integrase Mutants Primarily Defective for Integration, Not PIC Nuclear Import

2002 ◽  
Vol 76 (21) ◽  
pp. 10598-10607 ◽  
Author(s):  
Ana Limón ◽  
Eric Devroe ◽  
Richard Lu ◽  
Hina Z. Ghory ◽  
Pamela A. Silver ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Cell Nuclei ◽  
Immunodeficiency Virus ◽  
Viral Cdna ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome. A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells. One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types. In this study we confirmed that HIV-1V165A and HIV-1R166A were replication defective and that less mutant viral cDNA localized to infected cell nuclei. However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import. First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei. Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1V165A and HIV-1R166A, suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses. Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli. Based on these results, we conclude that HIV-1V165A and HIV-1R166A are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells.

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