We report here a positive impact of hyperthermia on HIV-1 replication. Hyperthermia is known to enhance the functions of immune cells and to confer protection against pathogen infection 
, Previous studies on temperature and HIV-1 mostly focused on chronically infected cell lines 
or used non-physiological heat shock treatment to study viral reactivation from latency (a few minutes at 42–45°C 
). Here, we report that elevation of temperature to fever-like levels (39.5°C) stimulates HIV-1 replication in primary CD4+ T lymphocytes as well as in Hela and Jurkat cell lines. In single-cycle infection assays, hyperthermia increased HIV-1 infection by 2 to 7 fold. This stimulation was apparently not due to unspecific alterations of cellular metabolism, since cell growth, viability, or surface levels of various molecules were not significantly affected by hyperthermia.
To get insight into how hyperthermia stimulates HIV-1 replication, we compared the efficiency of various steps of the viral life cycle at 37°C and 39.5°C. Viral entry and fusion, measured by the Vpr-β-lactamase assay, were similar at the two temperatures. Enzymes have a range of conditions of pH, salt concentration, and temperature, in which they display optimal activity. We did not observe an effect of hyperthermia on reverse transcriptase, as both in vitro
catalytic activity of the enzyme and the levels of viral DNA synthesis in infected cells were unchanged by temperature. We then examined the influence of temperature on the viral transcription step. Hyperthermia did not induce basal LTR activity without Tat. However, in the presence of Tat, hyperthermia lead to a significantly better transactivation of the LTR. This is in line with earlier reports, demonstrating that a transient heat shock at 42°C increases HIV-1 transcription in monocytic cells lines 
. Noteworthy, the activity of the CMV promoter was not increased at 39.5°C (not shown), suggesting that hyperthermia does not trigger a global increase of cellular transcription. Accordingly, the steady state levels of several cellular proteins (actin, CD4, ICAM-1, MHC-I, etc.) were apparently similar at normal and elevated temperatures.
To characterize the molecular mechanism by which hyperthermia up-regulates HIV-1 infection and transcription, we examined the role of Hsp90. This protein exerts diverse functions in normal and stressed cells, through its ATPase activity and its protein binding domain 
. It acts as a chaperone for many cellular proteins. Hsp90 assists folding, assembly, intracellular transport, maintenance and degradation of proteins, and regulates cell signaling and cell cycle 
. Hsp90 is involved in HIV-1 infection at 37°C, regulating viral gene expression 
. Hsp90 also impacts the replication of other viral species, such as Human Cytomegalovirus, Influenza Virus, Flock House Virus and Hepatitis C Virus 
. We show here that the levels of Hsp90 are augmented at 39.5°C, in primary lymphocytes and other cells ( and not shown). By using an immunofluorescence technique allowing the visualization of nascent viral RNA in living cells, we demonstrate that, in presence of Tat, Hsp90 can be found in the nucleus, at HIV-1 transcription sites. This localization was rather infrequent at 37°C, but was significantly increased at 39.5°C (27% and 70% co-localization, respectively). Furthermore, 17-AAG, a pharmacological inhibitor of Hsp90, reversed the stimulating effect of hyperthermia on single-cycle infection in P4C5 cells. Altogether, these results point out for a previously uncharacterized role of Hsp90, facilitating HIV-1 transcription and replication at 39.5°C. It will be worth further dissecting how Hsp90 acts on viral transcription at this temperature. One can speculate that the chaperone protein may bind more efficiently to the P-TEFb/Tat/TAR transcription complex 
and thus increase its activity, and/or may enhance chromatin modeling and accessibility to the viral promoter 
Mechanisms regulating HIV-1 gene expression are also involved in viral reactivation from latency 
. We show here that the conditioned medium from PBMCs induced viral reactivation, in the J-Lat 10.6 model of latently infected T cells. Strikingly, reactivation was more pronounced at 39.5°C than at 37°C. Futures studies will help understanding which cytokines or other molecules produced by PBMCs mediate this effect. For instance, heat shock at 42°C is known to act in synergy with IL-6 to induce viral reactivation in a latently infected monocytic cell-line 
. It will be of interest to compare the stimulating effect of IL-6 and other cytokines, at normal and fever-like temperatures, not only in J-Lat cells, but also in other models of viral latency (PBMCs from HAART-treated patients, or latently-infected, resting primary CD4+ T cells 
In this study, we have focused our analysis of the effect of temperature on a few key steps of the viral life cycle. We demonstrate that hyperthermia globally facilitates viral replication. At 39.5°C, viral entry, fusion and reverse transcription occur normally, whereas Tat-mediated transactivation of the LTR is significantly more efficient. It has been previously reported that the activity of HIV-1 integrase and protease is not increased at 39.5°C 
. This does not rule out the possibility that other steps of HIV-1 infection (nuclear import, selection of integration sites in the cellular genome, viral translation, assembly, release, etc.) might be positively or negatively modified at a fever-like temperature.
What is the physiological relevance of our observations? Patients treated with HAART and with controlled viremia can experience transient bursts of HIV-1 replication termed viral blips 
. Furthermore, co-infections are frequent in HIV-1-positive individuals and are often associated with fever and acute illnesses 
. For instance, Plasmodium falciparum
, the causative agent of malaria, induces recurrent, strong episodes of fever lasting 2–3 days, which correlates with increased viral loads 
. The origin of these viral blips, or of other more pronounced viral rebounds is likely multi-factorial. Our results suggest that fever may directly stimulate viral replication or reactivation from latent reservoirs, in association with other inflammatory or immunological events.