All known primate lentiviruses contain one or two genes (vpr
) that are considered vpr
homologs. The various genes in the vpr
family are structurally related because they have predicted amino acid sequence homology and encode products that are virion-encapsidated (54
). These genes are involved in multiple aspects of the biology of primate lentiviruses, although their precise roles in viral replication and modulation of host functions are still unclear.
We and others previously reported that species-specific factors modulate the induction of G2
arrest by members of the vpr
). SIVagm and SIVsyk vpr
genes are capable of arresting African green monkey kidney cells but are unable to do so in human cells. In contrast, HIV-1, HIV-2, and SIVsm vpr
genes function in both simian and human cell types, although SIVsm Vpr functions more efficiently in simian cells than it does in human cells. These differences could not be explained on the basis of differential protein stability or subcellular localization (51
). The species-specific cell cycle arrest differences between SIVagm and HIV-1 Vpr proteins indicate that these proteins may signal through cellular pathways that are divergent among primates.
We first compared vprAGM
at the level of transactivation. We wished to ascertain whether transactivation by vprAGM
is species specific, as is its ability to induce G2
arrest. Surprisingly, vprAGM
was able to induce transactivation in human cells, despite its inability to induce cell cycle alteration in these cells. This observation suggests that vprAGM
may exert transactivation by a mechanism that is, at least in part, independent of cell cycle manipulation. To corroborate this observation, we resorted to the use of caffeine, a known inhibitor of vpr
) and DNA damage-induced (36
arrest. Caffeine potently inhibited the cell cycle arrest by vprAGM
in African green monkey kidney cells but produced little or no inhibition of its transactivation effect in either monkey or human cells.
The transactivation effects reported here and elsewhere for the various primate vpr
genes are modest compared with the transactivation effects exerted by other, more classical viral transactivators (e.g., HIV-1 tat
or human T-cell leukemia virus tax
). The importance of vpr
-induced transactivation, however, is underscored by two observations. First, HIV-1, HIV-2, SIVmac, and SIVagm vpr
genes are able to transactivate their respective LTRs (39
). This observation establishes the conservation of this function through evolution. Second, experiments in which the abilities of tat
to transactivate were tested in parallel indicated that they were not mutually exclusive but were synergistic (28
). Thus, coexpression of the two genes provided a multiplicative effect on the promoter activity of the LTR.
We also wished to examine whether the ability of vprHIV-1 to cause apoptosis would be paralleled by vprAGM. Indeed, vprAGM is capable of inducing apoptosis in African green monkey kidney cells. This induction of apoptosis was not observed when vprAGM was expressed in human cells, and therefore apoptosis appears be determined by species-specific factors.
Abrogation of the cell cycle effect by caffeine treatment suppressed the levels of apoptosis by vprAGM
, as it did for vprHIV-1
. The potential link between the induction of G2
arrest and the onset of apoptosis is likely to be complex, since multiple signaling connections have been proposed. Perhaps the area of radiation-induced DNA damage has produced the clearest results to date. DNA damage is a natural signal that may induce cell cycle arrest and apoptosis. Cell cycle arrest following cellular insult allows for the repair of damaged DNA to protect the organism from the repercussions of mutation (21
). Cell cycle block can occur before DNA replication, at the G1
/S checkpoint, or before chromosome segregation, at the G2
/M checkpoint (21
). Many DNA-damaging agents, including certain antineoplastic drugs, exert their effect at the G2
/M phase (9
) and, ultimately, commit the cell to death (32
). The effects of vpr
, specifically the G2
block, may be mediated along similar pathways to the effects of genotoxic agents (43
It was initially thought that the cell cycle perturbation function of vprHIV-1
would require that the protein be localized in the nucleus. This was supported by experiments by Di Marzio et al. (15
), who failed to find vprHIV-1
mutants that could cause G2
arrest in the absence of nuclear localization. Later mutagenesis experiments demonstrated that nuclear localization was not a requisite for vprHIV-1
to function as a cell cycle inhibitor (33
). We tested whether the subcellular localization of vprAGM
might explain the species specificity of the effects of this protein. We found that in both human and African green monkey kidney cells a GFP-vprAGM
fusion protein is also found in the nucleus. Thus, subcellular localization does not explain the inability of vprAGM
to cause G2
arrest and apoptosis in human cells.
Our results point out important functional differences between the HIV-1 and SIVagm vpr genes (Fig. ). HIV-1 vpr appears to induce G2 arrest as a primary effect, whereas transactivation and apoptosis appear to be downstream effects of G2 arrest. Thus, inhibition of G2 arrest will abrogate such downstream effects. SIVagm vpr induces these three effects independently. Thus, abrogation of vprAGM-induced G2 arrest has no effect on the onset of apoptosis or transactivation of the LTR.
FIG. 9 Proposed model to summarize the relationships among various functions of vprHIV-1 and vprAGM. The observed effects of drugs are depicted in grey. Caffeine inhibits vprHIV-1 G2 arrest and also its downstream effects, apoptosis and transactivation. For (more ...)
Taken together, our results suggest that while the multiple functions of vpr are conserved among various primate lentiviruses, the mechanisms leading to the execution of such functions are divergent.