In this report, we provide evidence that Hsf1 is the main regulator responsible for Hsp16 elevation and anti-Vpr responses in fission yeast cells. The fact that Hsf1 is responsible for Hsp16-mediated response to Vpr indicates that the effect of Hsp16 on Vpr resembles the cellular heat shock responses. Indeed, overexpression of hsf1 completely reduced Vpr'-induced cell cycle G2 arrest as shown by reversion of the cell elongation (Fig. 3A - d vs. b), shift of the cellular DNA content from G2 to G1 (Fig. 3A - b) and additional heat treatment of hsf -expressing cells did not significantly enhance the suppressive effect of Hsf1 on Vpr (Fig. 3B - h vs. d).
Even though vpr gene expression triggers Hsp16 elevation, Vpr appears to prevent further elevation induced by heat treatment (Fig. 5A - e; Fig. 5B - a, lane 4; Fig. 5B - b, lane 5), suggesting a counteracting effect of Vpr on heat stress-like cellular response possibly through transcriptional regulation of hsp16. This notion is supported by our observations that induction of Hsp16 by heat treatment failed to counteract Vpr-induced cell death (Fig. 4A -3, bottom plate). However, overexpression of hsp16 under the control of an exogenous nmt1 promoter completely suppressed Vpr-induced cell death under the same heat shock conditions (; Fig. 4A -4). Possible transcriptional down-regulation of hsp16 by Vpr is further evidenced by the results shown in Fig. 5A, in which a GFP reporter was fused with the endogenous hsp16 promoter  and expression of vpr eliminated the Hsp16 elevation (Fig. 5A - e, h). Although the molecular mechanism underlying this transcriptional suppression of hsp16 is unclear at the moment, the fact that Hsf activates Hsps through binding of the Hsp promoters  and overexpression of hsf1 or hsp16 through an exogenous adh or nmt1 promoter alleviates the Vpr activity (Fig. 3A; ) support the idea that Vpr may affect expression of hsp16 through competition with Hsf for control of hsp16 expression. One possible scenario is that Vpr may inhibit Hsf1 that results in reduced transcription of hsp16. Alternatively, since Vpr is a weak transcriptional activator through binding to the transcriptional factor Sp1 , it is also possible that Vpr may compete with Hsf1 by binding to the Sp1 region of the hsp16 promoter. Obviously additional tests are needed to elucidate these possibilities. Interestingly, only the wild type Vpr was able to inhibit Hsp16 at early hours (23 hrs) after induction, as a single amino acid substitution from phenylalanine to isoleucine at position 34 of Vpr attenuated its ability to suppress the increase of Hsp16 after acute heat shock (Fig. 5A - i vs. h; Fig. 5B, lane 6 vs. lane 4). In fact, an even higher level of Hsp16 was observed. This is presumably due to the inability of Vpr' to compete with Hsf-mediated Hsp16 elevation. Thus, assuming that expression of hsp16 is responsive to both the presence of Vpr and heat shock treatment, this larger increase of Hsp16 could be an additive effect. Since amino acid substitution at residue 34 of Vpr diminishes the ability of Vpr to induce cell death but retains induction of G2 arrest [20, 28, 29], a plausible possibility is that suppression of Hsp16 and induction of cell death by Vpr share common pathways.
It should be mentioned that whether Vpr-induced G2 arrest and cell death are two functionally independent activities is still of debate. Earlier reports suggested that these two activities are separable both in fission yeast and mammalian cells [20–24]. However, recent reports indicated Vpr-induced apoptosis is cell cycle dependent [25, 26]. Although reasons for these discrepancies are not completely clear at the moment, it is noticed that apoptosis shown in the Andersen's study describes a late event as cells were collected 48–72 hrs after viral infection . Prolonged cell cycle G2 arrest results in apoptosis. Thus, it is not surprising to find that apoptosis described in the Andersen's study is ANT-independent and ANT-dependent apoptosis was documented previously . Additional difference between the apoptosis described by Andersen et al from others is also noticed in the examination of two Vpr mutations. The R77Q and I74A mutants, which separate the apoptosis and G2 arrest induced by Vpr [24, 54], showed no separation between the G2 induction and apoptosis. In our study, the F34IVpr mutant is unable to induce cell death but retains its ability to induce cell cycle G2 arrest both in fission yeast [21, 27, 28] and mammalian cells (; our unpublished data) It thus allowed us to differentiate the effect of a wild type Vpr vs. a mutant Vpr that only confers the inhibitory effect on cell cycle regulation.
Responsive elevation of fission yeast Hsp16 and its human paralogue HSP27 (our unpublished data) suggests that the cellular heat stress-like responses might be antagonistic to Vpr. Indeed, we previously showed that overexpression of hsp16 and human HSP27 suppress the Vpr activities, including cell cycle G2 arrest and cell killing, both in fission yeast and human cells (; our unpublished data). However, the suppressive effect of yeast Hsp16 and human HSP27 on Vpr are not identical. Overproduction of Hsp16 completely eliminated all of the Vpr activities including the positive role of Vpr in supporting viral replication in macrophages (Fig. 2C - a). Under the same condition, however, HSP27 has no clear suppressing effect against Vpr in macrophages (Fig. 2C - b). One possible difference between these two HSPs is that Hsp16 associates directly with Vpr  but no clear HSP27-Vpr interaction was detected both in vitro and in vivo (our unpublished data). Unlike Hsp16, overexpression of HSP27 is unable to block nuclear transport capacity of Vpr (our unpublished data). Since nuclear transport of Vpr is required for HIV-1 infection in non-dividing cells such as macrophages [7, 55, 56], the inability of HSP27 to block nuclear import of Vpr could potentially explain why it has no effect on HIV-1 infection in macrophages.
There appears to be a dynamic interaction between vpr gene expression and activation of Hsp16 in fission yeast. Results of our parallel studies in mammalian cells indicated a similar dynamic and antagonistic interaction between Vpr and HSP27 (our unpublished data). This finding is not surprising because activation of heat shock proteins by Hsf1 is a highly conserved cellular process among all eukaryotic cells . All eukaryotes encode at least one heat shock factor that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, i.e., the heat shock element, that is absolutely conserved among eukaryotes . Based on the data presented, we hypothesize that expression of vpr or HIV infection elicits a transient activation of the small heat shock proteins (sHsps) of eukaryotes through an Hsf-mediated pathway. Activation of these sHsps is most likely a part of the cellular antiviral reaction to HIV infection and specifically to Vpr. However, these stress responses are normally not sufficient to suppress the Vpr activities because of active counteraction from Vpr. Importantly, however, the Vpr activities could be completely blocked when sHsp's are produced under control of an exogenous promoter thus avoiding transcriptional inhibition by Vpr. Since the Vpr-specific activities have been linked to such clinical manifestation of AIDS as activation of viral replication , suppression of host immune responses  and depletion of CD4+ T-lymphocytes [12, 58], this finding could potentially provide a new approach to reducing Vpr-mediated detrimental effects in HIV-infected patients by stimulating expression of sHsps.