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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Published on: August 31, 2014
Evidence for Vpr-dependent HIV-1 replication in human CD4+ CEM.NKR T-cells
Tao Zhou1, Ying Dang, Jacob J Baker
1Department of Microbiology and Molecular Genetics, Michigan State University, 567 Wilson Road, 2215, Biomedical and Physical Sciences Building, East Lansing, MI 48824-4320, USA.
Human immunodeficiency virus type 1 (HIV-1) replication in NKR T cells depends on the viral protein R (Vpr). Vpr enhances HIV-1 replication from the second cycle by overcoming an early-stage replication block, independent of DCAF1 and G2 arrest.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Viral protein R (Vpr) is a lentiviral protein crucial for primate lentivirus replication and pathogenesis.
- HIV-1 Vpr exhibits in vitro activities including G2 phase cell cycle arrest and enhanced viral replication.
- Human CD4+ CEM.NKR (NKR) T cells exhibit potent restriction of wild-type (WT) HIV-1 replication.
Purpose of the Study:
- To investigate the role of Vpr in HIV-1 replication within restricted NKR T cell lines.
- To compare the replication efficiency of wild-type, Vif-defective, Vpr-defective, and Vpu-defective HIV-1 in NKR cell clones.
Main Methods:
- Isolation and characterization of NKR cell clones with varying resistance levels (non-permissive, semi-permissive, permissive) to WT HIV-1.
- Comparative replication assays using different HIV-1 mutant viruses (WT, Vif-defective, Vpr-defective, Vpu-defective).
- Analysis of viral protein processing, virion infectivity, and the effect of arsenic trioxide (As2O3) and Vpr cofactor DCAF1 disruption.
Main Results:
- Vpr and Vif were essential for HIV-1 replication in non-permissive NKR cells, while Vpu was not required in permissive and semi-permissive cells.
- Vpr-defective HIV-1 produced infectious virions but showed reduced reverse transcription efficiency in the subsequent infection cycle.
- Arsenic trioxide restored WT HIV-1 replication in non-permissive cells, but not Vpr-defective virus replication; Vpr's enhancing activity was independent of DCAF1 and G2 arrest.
Conclusions:
- HIV-1 replication in NKR cells is critically dependent on Vpr.
- Vpr promotes HIV-1 replication starting from the second cycle by resolving an early-stage replication block.
- The Vpr-mediated enhancement of HIV-1 replication in NKR cells does not involve DCAF1 binding or G2 cell cycle arrest.
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