At the Conference on Retroviruses and Opportunistic Infections back in 2002, Michael Sherman presented the first evidence that the HIV-encoded protein Vpr arrests the cell cycle of infected CD4 T cells in vivo (Vpr has long been known to exert this effect in vitro in a wide variety of cells, even yeast). This data has now finally been published in the latest issue of the Journal of Virology. The researchers isolated HIV-infected cells from recently infected individuals using a technique involving staining for antibodies to p24, subsequently confirming infection status using DNA PCR. The ratio of particular types of DNA content was then analyzed to ascertain whether the cells were arrested in the G2 stage of the cell cycle, and abnormal DNA content consistent with G2 arrest was reproducibly found in infected (but not uninfected) cells from every study participant analyzed. The authors also note that a surprising number of CD4-negative T cells were found to be infected and arrested in G2; analyses showed that these cells expressed markers of normal lymphocytes, such as rearranged T-cell receptors, and were always negative for CD8, suggesting that they expressed CD4 at the time of infection but the receptor was then downregulated and not re-expressed (similar findings have been reported previously). The work of Sherman and colleagues is notable because, while a multiplicity of potential effects of HIV infection on CD4 T cells have been posited based on in vitro studies, confirmation that these effects actually occur in vivo is all too rare.
Journal of Virology, November 2006, p. 10407-10418, Vol. 80, No. 21
Erik S. Zimmerman, Michael P. Sherman, Jana L. Blackett, Jason A. Neidleman, Christophe Kreis, Pamela Mundt, Samuel A. Williams, Maria Warmerdam, James Kahn, Frederick M. Hecht, Robert M. Grant, Carlos M. C. de Noronha, Andrew S. Weyrich, Warner C. Greene, and Vicente Planelles
Received 9 June 2006/ Accepted 10 August 2006
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.