Article

Effects of mutant Vpr/Vpx on HIV-2 assembly demonstrated by immunoelectron microscopy.

Department and Institute of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan, ROC.
Proceedings of the National Science Council, Republic of China. Part B, Life sciences 02/2001; 25(1):50-8. pp.50-8
Source: PubMed

ABSTRACT The virion-associated accessory proteins Vpr and Vpx of human immunodeficiency virus type 2 (HIV-2) are required for efficient viral replication. Vpr could be important for Vpx assembly. To investigate the interaction of Vpr and Vpx with respect to the effects of reverse transcriptase (RT) activity, viral particle information and Vpx expression site directed mutagenesis was carried out to construct Vpr, Vpx and double Vpr/Vpx HIV-2 mutants. These mutants were used for infection of peripheral blood mononuclear cells (PBM), human acute lymphoblastomic leukaemia cells (CEM-CM3) and HeLa CD4+ cells. Visualization of Vpx expression was carried out using FITC and gold labelling by means of laser scanning confocal microscopy and semi quantitative immunoelectron microscopy. Intracellular and extracellular localizations of Vpx were determined by means of fine structural analysis. Up to 80-90% reduction in the RT activity, total number of viral particles, and average Vpx expression was observed after infection of target cells with the Vpr mutant strains. In addition, intracellular Vpx expression was reduced to 51.2% with the Vpr mutant. Only 0.02% Vpx expression was detected after mutation at amino acid 62. These results provide evidence that Vpr or Vpr/Vpx mutants reduce RT activity and interfere with the expression of Vpx in HIV-2 particles during viral assembly. Vpr is efficient for Vpx corporation during viral assembly.

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    Article: Knockdown of the cellular protein LRPPRC attenuates HIV-1 infection.
    Cameron J Schweitzer, John M Matthews, Christian J Madson, Meghan R Donnellan, Ronald L Cerny, Michael Belshan
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    ABSTRACT: HIV-1 exploits numerous host cellular pathways for productive infection. To identify novel factors involved in HIV-1 replication, HIV-1 integrase and matrix protein complexes were captured at 4 hours post infection for proteomic analysis using an affinity purification system. Leucine-rich PPR-motif containing (LRPPRC) protein, a cellular protein involved in mitochondrial function, cell metabolism, and cell-cycle progression was identified as one of the candidate HIV-1 factors. Co-immunoprecipitation RT-PCR experiments confirmed that LRPPRC associated with HIV-1 nucleic acids during the early steps of virus infection. To establish if LRPPRC was critical for HIV-1 infection, three independent LRPPRC knockdown cell lines were constructed (2.7, 3.6, and 4.1). Subcellular fractionation of these cell lines revealed differential knockdown of LRPPRC in subcellular compartments. LRPPRC was knocked down in the insoluble/cytoskeletal fractions of all three cell lines, but the 3.6 and 4.1 cells also showed a reduction in nuclear LRPPRC. Additionally, several cellular factors were downregulated and/or disrupted by loss of LRPPRC. HIV-1 infection was reduced in all three cell lines, but virus production and RNA encapsidation were unaffected, suggesting that LRPPRC was critical for the afferent stage of virus replication. Two of the three cell lines (3.6, 4.1) were refractory for murine leukemia virus infection, a virus dependent on cellular proliferation for productive infection. Consistent with this, these two cell lines exhibited reduced cellular growth with no loss of cellular viability or change in cell cycle phenotype. The early steps of virus infection were also differentially affected among the cell lines. A reduced level of preintegration complex formation was observed in all three cell lines, but viral DNA nuclear import was reduced only in the 3.6 and 4.1 cells. Combined, these data identify LRPPRC as a HIV-1 factor that is involved in HIV-1 replication through more than one mechanism.
    PLoS ONE 01/2012; 7(7):e40537. · 4.09 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

amino acid 62
 
average Vpx expression
 
double Vpr/Vpx HIV-2 mutants
 
efficient viral replication
 
HeLa CD4+ cells
 
HIV-2 particles
 
human acute lymphoblastomic leukaemia cells
 
human immunodeficiency virus type 2
 
intracellular Vpx expression
 
laser scanning confocal microscopy
 
peripheral blood mononuclear cells
 
semi quantitative immunoelectron microscopy
 
target cells
 
total number
 
viral particle information
 
viral particles
 
virion-associated accessory proteins Vpr
 
Vpr mutant strains
 
Vpr/Vpx mutants
 
Vpx expression site
 

J. J. Wan