Infection with PRRSV causes a high mortality and leads to substantial economic losses to the swine industry worldwide. Vaccines have been developed to control PRRSV infection, but failed to provide sustainable disease control because of the viral immune evasion strategies and the antigenic heterogeneity of field strains
. Hence, the development effective antiviral strategies to combat PRRSV infection is an urgent necessity. The most common antiviral strategy relies on directly inhibiting viral proteins. However, this strategy invariably results in the emergence of drug resistance, as the virus can readily mutate to circumvent inhibition due to the error-prone nature of the ploymerase, even under conditions of combinatorial therapy targeting multiple viral proteins
. Targeting cellular proteins for developing new generation of antivirals is gaining concern
[19–21, 32]. HSP90 inhibitors, such as herbimycin A, radicicol, GA and its derivatives, characterized as effective anti-cancer therapeutics and several of which now in phase I and II clinical trials
[33, 34], have been shown to have strong antiviral activity. Strikingly, in the case of poliovirus, GA treatment did not led to the emergence of drug resistance within 10 passages
. Therefore, it is thought that HSP90 inhibitors could be promising broad-range antiviral agents. We have previously shown that HSP90 was elevated in PRRSV infected lungs relative to uninfected negative control (UNC) lungs based on transcriptome and proteome approaches
[35, 36], suggesting that HSP90 might be an important host factor for PRRSV infection as observed for other viruses. Therefore, we tried to inhibit HSP90 and test whether the inhibition could affect PRRSV infection.
In the present study, we found both inhibitors could block the synthesis of PRRSV RNA, and thus reduce viral infection in vitro. PAMs are known to be the primary host cellular target for PRRSV replication, thus the significant antiviral effects of these agents in these cells suggests that they might also be effective inhibitors against PRRSV in vivo. But it remains to be determined. Notably, GA or 17-AAG treatment could not induce IFN-β gene expression in both cell types. A previous research has showed that GA can inhibit the dsRNA- or virus-induced IFN-β gene expression in HeLa cells
. These results suggest that the anti-PRRSV activities performed by HSP90 inhibitors are not due to the activation of interferon response.
We also evaluated the effects of siRNA-mediated knockdown of HSP90 on PRRSV infection. The simultaneous depletion of both proteins led to a dramatic reduction of viral infection. However, no significant inhibitory effects were observed when individual knockdown, suggesting that these two isoforms might have overlapping functions during PRRSV infection. Interestingly, HSP90α was up-regulated after transfection with siRNA targeting HSP90β (Figure,
6A, lane 4), which is consistent with a previous research
, indicating a compensatory up-regulation. But the corresponding augmentation of HSP90β after transfection of siHSP90α was not observed (Figure,
6A, lane 3), which may be due to the fact that HSP90β is generally constitutive and not sensitive to a great variety of stimuli
As known, HSP90 can be involved in different stages of the viral life cycle. Our results showed that the PRRSV RNA synthesis was prevented by GA and 17-AAG treatment, suggesting that HSP90 is somehow involved in supporting the PRRSV replication. In addition, lower levels of viral protein and viral production were found. Hence, it is not excluded that HSP90 may also be involved in the PRRSV life cycle at the steps of protein synthesis and budding. Notably, the inhibitory effects observed in this study are not due to inhibition in virus adsorption or entry since in all exprements, the inhibitors were added at 1 h.p.i, when PRRSV has been internalized in host cells
. The addition of inhibitors, even at 4 h.p.i, also showed significant inhibitory effects (data not shown). However, these results could not exclude the possibility that HSP90 could regulate PRRSV infection in absorption and internalization.
The exact roles that HSP90 plays during PRRSV infection remain to be determined. HSP90 can regulate viral infection by modulating the host processes or interacting with viral proteins directly
[21, 25]. Therefore, further study will be mainly performed in our laboratory in two aspects: (I) identification of PRRSV protein associated with HSP90 directly; (II) investigation whether HSP90 is exploited by PRRSV to regulate cellular processes for its benefit.