Cell killing via direct viral infection is one mechanism underlying the CD4+
T-cell depletion that occurs during HIV-1 infection. The mechanisms by which the virus-encoded proteins can cause cytopathicity have been described in great detail using in vitro models. However, few studies confirm these virus-dependent phenotypes in vivo or even in primary CD4+
T cells infected with full-length HIV-1 in vitro. It has previously been shown that Vpr expression is necessary and sufficient to activate the G2
checkpoint when Vpr is expressed in transformed cell lines and activated CD4+
T lymphocytes (23
). Such arrested cells ultimately die as a result of apoptosis (4
). In the present study, we demonstrate that anti-p24Gag
-immunoreactive, activated CD4+
T cells from HIV-1-infected individuals are arrested in G2
. We also report Vpr-dependent G2
arrest in primary CD4+
T cells infected in vitro with HIV-1 molecular clones. Furthermore, in these primary CD4+
T cells, G2
arrest was relieved by RNA interference-mediated knockdown of ATR.
We also extended our studies to another physiologically relevant target cell for HIV-1 infection, the macrophage. As expected, Vpr did not alter the DNA content of primary monocyte-derived macrophages, due to their nondividing status. To our surprise, however, HIV-1 Vpr failed to induce phosphorylation of the known ATR targets, H2AX and Chk1. For H2AX, we demonstrated that the defect in MDM is at the level of ATR signaling and not H2AX itself, since an ATM-activating stimulus results in H2AX phosphorylation and focus formation. We found that the failure to activate ATR in MDM was due to the lack of protein expression of at least three essential proteins in the signaling axis: ATR, Chk1, and Rad17. Based on these findings and on our previous report that Vpr-induced apoptosis is ATR dependent (4
), we hypothesize that macrophages are refractory to Vpr-induced apoptosis. If confirmed, the predicted resistance to Vpr-induced apoptosis could be one contributing factor to the ability of macrophages and, perhaps, other postmitotic cells to serve as viral reservoirs. Sensitizing postmitotic cells to the cytopathic properties of Vpr by reactivating the ATR pathway could provide a means to eliminate these reservoirs.
The apparent absence of ATR in quiescent lymphocytes was previously reported by Jones et al. (28
), and we herein report, for the first time, that ATR is also absent in MDM. The absence of ATR in the previous two cell types is surprising in light of previous experiments that demonstrate an absolute requirement for ATR during mouse development (8
). In addition, experiments with a conditionally deleted form of the ATR gene indicated that it is essential for the viability of somatic cells (11
). Therefore, the regulation of ATR gene expression and its relationship with the dividing status of cells deserve further investigation.
Virion-bound Vpr is required for efficient infection of nondividing cells, including monocyte-derived macrophages (9
). Since induction of G2
arrest and infection of nondividing cells are independent separable functions of Vpr (13
), our results do not negate an immediate-early role of Vpr in facilitating infection of nondividing cells.
ATR is thought to be a sensor of DNA replication stress through binding to RPA-rich ssDNA regions in the genome (10
). To test whether Vpr activates ATR in a canonical manner, we assayed for the presence of RPA-rich foci and RPA phosphorylation in the context of HIV-1 infection. Our results conclusively demonstrate that HIV-1 infection of primary lymphocytes induces DNA replication stress and that removal of vpr
from the virus genome relieves this effect.
To ascertain whether the ability of Vpr to induce G2 arrest can be observed in vivo, we performed experiments with human samples. We first observed that in vitro culture of lymphocytes from an infected patient would result in virus amplification and then induction of G2 arrest and RPA foci. This experiment indicates that a primary virus, which is not molecularly or biologically cloned and is, therefore, presumably a quasispecies, maintains the cell cycle-perturbing properties previously ascribed to Vpr in overexpression systems.
In a second experiment with in vivo samples, freshly isolated CD4+ lymphocytes from recent seroconverters were interrogated for DNA content in the absence of any in vitro treatment, other than cell sorting. Our results show, for the first time, that p24Gag+ lymphocytes, but not p24Gag− ones from the same patient, can be found arrested in G2. We observed this for all patients from whom sufficient p24Gag+ cells could be recovered.
It is somewhat surprising that the CD4-negative compartments from the in vivo samples showed levels of infection similar to those of the CD4-positive compartment (Table ). This infected CD4−
population likely represents HIV-1-infected cells that have downregulated CD4 surface expression due to expression of Nef and other viral gene products (reviewed in reference 35
). There is strong precedent for the existence of significant levels of infected cells in the CD4-negative compartment. In a study by Marodon et al. (38
), HIV-1 DNA copies were analyzed by quantitative PCR in sorted CD4+
, and CD4−
lymphocytes from HIV-infected individuals. In this study, high levels of viral DNA were found in 16 out of 19 patients analyzed. When the surface phenotypes of these cells were analyzed, they were shown to contain typical markers of normal lymphocytes, such as rearranged T-cell receptors, and were always negative for CD8. These results suggests that infected cells were originally CD4+
and had efficiently downregulated CD4 following infection.
The levels of detected viral DNA copies (two to seven HIV-1 Gag DNA copies per p24Gag+
cell) we report (Table ) are higher than we originally anticipated. Since the frequency of HIV-1-infected cells in peripheral blood is typically low, we expected viral DNA copy numbers in the vicinity of 1 per cell. A previous study, however, found that HIV-1-infected human splenocytes harbored integrated viral DNA copies that ranged from one to eight copies per infected splenocyte, with a mean around three, as evidenced by fluorescence in situ hybridization (30
). Jung et al. have in fact argued that copy numbers higher than 1 would help explain the high frequencies of in vivo recombination (30
In separate studies, we have shown that the ATR signaling cascade is required for Vpr-induced apoptosis (4
). Thus, we hypothesize that the attenuated cytopathicity of HIV-1 in macrophages may be explained, in part, by the failure of Vpr to activate ATR in macrophages. Several other questions, which compel further investigation, are raised by our findings. What is the precise molecular mechanism by which Vpr causes DNA replication stress? How are the expressions of ATR, Rad17, and Chk1 extinguished in postmitotic noncycling cells and stimulated in cycling cells? Will reconstitution of the ATR pathway in macrophages or other nondividing cells sensitize them to HIV-1-induced cytopathicity?