-
[show abstract]
[hide abstract]
ABSTRACT: Unintegrated human immunodeficiency virus (HIV) DNA are viral DNA products formed naturally during HIV replication. While the integrated proviral DNA form is transcriptionally active and results in productive infection, unintegrated DNA is also capable of expression of viral RNA and proteins. Previously, we showed that HIV Vpr enhances expression from integrase-defective HIV. Here we show that Vpr activation of expression is partially dependent upon the presence of a transcriptionally active HIV promoter and results in increased transcription of unspliced gag and spliced nef viral RNA. While Tat is detectable during infection with integrase-defective HIV, Tat levels are not affected by the presence of Vpr. Mutation studies reveal that Tat is dispensable for the Vpr-mediated enhancement of expression from unintegrated DNA. We find that virion-associated Vpr is sufficient for Nef expression from unintegrated viral DNA, resulting in the efficient downregulation of CD4 from the surface of infected cells. These results provide a mechanism by which Nef expression from unintegrated HIV type 1 DNA expression occurs.
Journal of Virology 11/2007; 81(19):10515-23. · 5.40 Impact Factor
-
Dong Sung An,
Robert E Donahue,
Masakazu Kamata, Betty Poon,
Mark Metzger,
Si-Hua Mao,
Aylin Bonifacino,
Allen E Krouse,
Jean-Luc Darlix,
David Baltimore,
F Xiao-Feng Qin,
Irvin S Y Chen
[show abstract]
[hide abstract]
ABSTRACT: RNAi is a powerful method for suppressing gene expression that has tremendous potential for therapeutic applications. However, because endogenous RNAi plays a role in normal cellular functions, delivery and expression of siRNAs must be balanced with safety. Here we report successful stable expression in primates of siRNAs directed to chemokine (c-c motif) receptor 5 (CCR5) introduced through CD34+ hematopoietic stem/progenitor cell transplant. After hematopoietic reconstitution, to date 14 months after transplant, we observe stably marked lymphocytes expressing siRNAs and consistent down-regulation of chemokine (c-c motif) receptor 5 expression. The marked cells are less susceptible to simian immunodeficiency virus infection ex vivo. These studies provide a successful demonstration that siRNAs can be used together with hematopoietic stem cell transplant to stably modulate gene expression in primates and potentially treat blood diseases such as HIV-1.
Proceedings of the National Academy of Sciences 09/2007; 104(32):13110-5. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The goal of this study was to develop a small-animal model to study human immunodeficiency virus type 1 (HIV-1) pathogenesis in blood and primary and secondary lymphoid organs. Rag2(-/-)gamma(c)(-/-) mice that are neonatally injected with human CD34(+) cells develop a functional human immune system (HIS), with human hematopoietic cells being found in the thymuses, peripheral blood, spleens, and bone marrow of the animals (hereafter these animals are referred to as HIS-Rag2(-/-)gamma(c)(-/-) mice). HIS-Rag2(-/-)gamma(c)(-/-) mice were infected with small amounts of CCR5-tropic HIV-1. Viral replication and immunophenotypic changes in the human cells in peripheral blood and lymphoid organs were examined. The productive infection of human cells in peripheral blood, thymus and spleen tissue, and bone marrow was detected. Ratios of CD4(+) T cells to CD8(+) T cells in the infected animals declined. Although no specific anti-HIV-1 immune responses were detected, immunoglobulin M (IgM) and IgG antibodies to an unidentified fetal calf serum protein present in the virus preparation were found in the inoculated animals. Thus, we have shown that the HIS-Rag2(-/-)gamma(c)(-/-) mouse model can be used for infection with low doses of CCR5-tropic HIV-1, which is most commonly transmitted during primary infections. HIS-Rag2(-/-)gamma(c)(-/-) mice can serve as a small-animal model for investigating HIV-1 pathogenesis and testing potential HIV-1 therapies, and studies with this model may replace some long and costly studies with nonhuman primates.
Clinical and Vaccine Immunology 05/2007; 14(4):391-6. · 2.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Live attenuated HIV vaccines offer a means to introduce exogenous sequences into the viral genome to target the virus elimination in vivo. Foreign genes inserted into the nef region of HIV-1 NL4-3 were found to be rapidly deleted following virus infection and/or replication, in a size dependent manner, in the human fetal Thymus/Liver implants of severe combined immunodeficient mouse (SCID-hu) model. When the murine heat stable antigen (HSA) of 283 bp was substituted into HIV-1 nef region, the viral loads in vivo were comparable to the negative control nef attenuated HIV-1, and the reporter HSA gene was not deleted upon infection. However, the murine Thy1.2 gene (505 bp) substituted into the nef attenuated HIV-1, upon infection and replication, deleted 441 bp in vitro and 437 bp in vivo, of the inserted Thy1.2 gene. When the enhanced green fluorescence protein (eGFP) gene (720 bp) was substituted for nef, virus replication was aborted in vivo in the Thy/Liv implants, as seen by the background levels of viral loads, comparable to mock infected implants, and the eGFP gene was deleted. When the herpes simplex virus thymidine kinase gene, HSV-TK (1.15 kbp), or HSA gene, was substituted into the viral vpr gene, TK but not HSA gene was deleted, upon infection in vitro. Moreover, NL-TKI reporter virus with both intact nef and vpr genes shows deletion of TK gene both in vitro and in vivo. Excision of foreign genes occurred within the exogenous segments but not in the viral own regions. These results suggest that larger "suicide" genes introduced via HIV-1 can be deleted upon infection. However, smaller size nucleotide sequences or genes (approximately 300 bp) inserted in place of viral nef or vpr gene may be used to target the virus or its components, for attack and elimination in vivo, and thus have implications for the development of live attenuated HIV vaccines.
Current HIV Research 11/2005; 3(4):377-92. · 1.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Retroviral DNA synthesized prior to integration, termed unintegrated viral DNA, is classically believed to be transcriptionally inert and to serve only as a precursor to the transcriptionally active integrated proviral DNA form. However, it has recently been found to be expressed under some circumstances during human immunodeficiency virus type 1 (HIV-1) replication and may play a significant role in HIV-1 pathogenesis. HIV-1 Vpr is a virion-associated accessory protein that is critical for HIV-1 replication in nondividing cells and induces cell cycle arrest and apoptosis. We find that Vpr, either expressed de novo or released from virions following viral entry, is essential for unintegrated viral DNA expression. HIV-1 mutants defective for integration in either the integrase catalytic domain or the cis-acting att sites can express unintegrated viral DNA at levels similar to that of wild-type HIV-1, but only in the presence of Vpr. In the absence of Vpr, the expression of unintegrated viral DNA decreases 10- to 20-fold. Vpr does not affect the efficiency of integration from integrase-defective HIV-1. Vpr-mediated enhancement of expression from integrase-defective HIV-1 requires that the viral DNA be generated in cells through infection and is mediated via a template that declines over time. Vpr activation of expression does not require exclusive nuclear localization of Vpr nor does it correlate with Vpr-mediated cell cycle arrest. These results attribute a new function to HIV-1 Vpr and implicate Vpr as a critical component in expression from unintegrated HIV-1 DNA.
Journal of Virology 05/2003; 77(7):3962-72. · 5.40 Impact Factor
