ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.
Subject(s)
DNA Replication , Gene Products, vpr/physiology , HIV-1/pathogenicity , Ataxia Telangiectasia Mutated Proteins , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/virology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/physiology , Cell Line , Cells, Cultured , Cytopathogenic Effect, Viral , DNA, Viral/biosynthesis , DNA, Viral/genetics , G2 Phase , HIV Infections/metabolism , HIV Infections/pathology , HIV Infections/virology , HIV-1/genetics , HIV-1/physiology , Humans , In Vitro Techniques , Macrophages/metabolism , Macrophages/virology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , RNA Interference , Signal Transduction , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
CONTEXT: Transmission of multiclass drug-resistant human immunodeficiency virus type 1 (HIV-1) may increase with wider use of antiretroviral therapy. OBJECTIVE: To determine trends in prevalence of HIV-1 drug resistance among recently infected individuals in a geographic area with a high penetration of antiviral treatment. DESIGN, SETTING, AND PATIENTS: Consecutive case series of 225 patients referred to a San Francisco, Calif, hospital with recent HIV-1 infection from June 1996 through June 2001. MAIN OUTCOME MEASURE: Time trends in the prevalence of genotypic and phenotypic primary drug resistance. RESULTS: Mutations associated with resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs) steadily increased from 0% in 1996-1997 to 12 (13.2%) in 2000-2001 (P =.01). There was 1 mutation associated with protease inhibitor resistance in 1996-1997 (2.5%) and there were 7 (7.7%) in 2000-2001 (P =.25). Genotypic resistance to nucleoside reverse transcriptase inhibitors (NRTIs) initially decreased and then returned to prior levels (P =.007 for test of homogeneity). Genotypic resistance to 2 or more classes of drugs increased from 1 (2.5%) to 12 (13.2%) (P =.004), but only 1 infection (1.2%) in the latter period was resistant to all 3 classes of agents (P =.58). Primary phenotypic resistance decreased for NRTIs from 21% to 6.2% (P =.03) and increased for NNRTIs from 0 to 8 (9.9%) (P =.02). Phenotypic resistance increased for protease inhibitors from 2.6% to 6.2% (P =.32). Median time to virologic suppression (<500 copies/mL) during therapy was 12 weeks for patients with genotypic evidence of resistance compared with 5 weeks for patients with drug-sensitive infections (P =.02). CONCLUSIONS: The frequency of primary resistance to NNRTIs is increasing, although resistance to all available classes of antiretroviral therapy remains rare. Genotypic resistance testing in recently infected persons predicts time to viral suppression during therapy.
