Pamela Mundt

Gladstone Institutes

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Warner C. Greene

Gladstone Institutes

Jason Neidleman

Gladstone Institutes

Michael P. Sherman

University of Missouri

Samuel A. Williams

Arsenal Biosciences (United States)

Carlos M. C. de Noronha

Albany Medical Center Hospital

Christophe Kreis

Gladstone Institutes

Frederick Hecht

University of California, San Francisco

Andrew S. Weyrich

University of Utah

Jason Hataye

National Institute of Allergy and Infectious Diseases

Robert M. Grant

Gladstone Institutes

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Gladstone Institutes

University of California, San Francisco

Institute of Virology of the Slovak Academy of Sciences

University of Utah

University of California, Los Angeles

University of California System

San Francisco General Hospital

Bipar

University of California, San Diego

National Institutes of Health

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Human Immunodeficiency Virus Type 1 Vpr Induces DNA Replication Stress InVitro and In Vivo

Journal of Virology (2006)

Erik S. Zimmerman Michael P. Sherman Jana Blackett Jason Neidleman Christophe Kreis

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G 2 . 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 G 2 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-p24 Gag -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 G 2 arrest.

S phase

10.1128/jvi.01212-06

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Nuclear Export of Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type 1 in Tissue Macrophages

Journal of Virology (2003)

Michael P. Sherman Carlos M. C. de Noronha L. Eckstein Jason Hataye Pamela Mundt

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.

Nuclear export signal

Nuclear pore

10.1128/jvi.77.13.7582-7589.2003

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Citations (43)