PRRSV infection results in substantial economic losses to the swine industry worldwide. However, no effective countermeasures exist to combat this deadly viral infection so far. The identification of host factors and exploration of their functions during virus infection not only will enable greater insight into the molecular mechanisms of viral pathogenesis, but also will provide a potential for the development of antiviral strategies.
Virus infection leads to changes of many host proteins expression, and up-regulation of HSP70 following viral infection has been widely observed . Recently, HSP70 was also found to be elevated after PRRSV infection based on transcriptome and proteome approaches [27, 30, 31]. In this study, we observed that PRRSV infection induced HSP70 expression in vitro (Figures 1 and 8C), implying that HSP70 may play a potential role in PRRSV infection.
Virus-induced HSP70 could be utilized to facilitate viral infection or to enhance intracellular defense against the invading microorganism. Hence, HSP70 can regulate the viral infection positively or negatively [32–34]. To better understand the role of HSP70 during PRRSV infection, we modulated the expression of HSP70 and analyzed the effect on viral infection. We observed that the down-regulation of HSP70 significantly reduced the level of viral N protein and viral production (Figures 2C, 3, 4, 5 and 8). PAMs are known to be the primary host cellular target for PRRSV replication, thus the significant anti-PRRSV effect of quercetin in these cells (shown in Figure 8) suggests that it might also be effective agent against PRRSV infection in vivo. However, overexpression of HSP70 following heat shock treatment resulted in slight increase of viral protein level and viral production (Figures 2C, 3A and 5), which is consistent with a previous research . This is likely due to the fact that PRRSV infection induced a rather high level of HSP70, which is sufficient to support PRRSV replication.
As expected, previous heat shock treatment could attenuate the inhibitory effects of quercetin on the PRRSV (Figure 4). Notably, quercetin at the concentration of 100 μM still has a strong inhibitory effect even with the previous heat shock treatment (Figure 4). This is likely because that quercetin at higher concentration powerfully inhibits the HSPs protein synthesis, and up-regulation of inducible HSPs (including HSP70) induced by previous heat shock treatment can not compeletly compensate the inhibition effect of quercetin. These results suggested other chaperones which are generally constitutive and not sensitive to stimuli may also be involved in the PRRSV life cycle, such as heat shock cognate protein 70 (HSC70) and HSP90β [35, 36]. HSC70 can be involved in different steps of viral life cycle, such as entry [37, 38], disassembly , translocation  and release . HSP90β, a constitutive cytoplasmic isoform of HSP90, has been reported as a critical host factor required for Japanese encephalitis virus (JEV) infectivity in BHK-21 cells . Further studies may be required to address whether HSC70 and HSP90β are involved in the PRRSV life cycle and to figure out their role during viral infection.
Using siRNA-mediated silencing approach, we specifically established the importance of HSP70 during PRRSV infection. We observed that siRNA-mediated depletion of HSP70 led to inhibition of viral protein synthesis and viral production in a dose-dependent manner. However, this inhibition could be rescued by heat shock treatment following transfection (Figure 5). These results indicated that HSP70 is essential for PRRSV infection, suggesting its proviral nature.
Positive-sense RNA virus infection forms dsRNA RI following the synthesis of complementary negative-sense RNA which is used as template to synthesize new strands. To investigate whether HSP70 play any role in the PRRSV replication, we detected the dsRNA level using specific antibody (J2). Our results showed that the knockdown of HSP70 resulted in reduction of dsRNA (Figure 6), indicating HSP70 is important for PRRSV replication. Viral dsRNA is contained in the RTC, hence dsRNA is used as a marker to examine the formation of viral RTC [5, 6, 28, 43, 44]. The reduction of dsRNA level may be due to the blockade of viral RTC formation. Confocal microscopic analysis was performed to examine if HSP70 associates with the RTC. We observed a strong colocalization of cytoplasmic HSP70 and dsRNA in PRRSV-infected cells (Figure 7), suggesting HSP70 may be involved in the formation of viral RTC and thus affect the viral replication. The formation of RTC composed of viral dsRNA RIs, viral replicases, altered cellular membranes and some cellular proteins, is a hallmark of all positive-stranded RNA viruses . HSP70 is frequently recruited to help the assembly of viral replicases into the RTC [23–26, 45]. Previous studies have implicated that several replicases of PRRSV, including NSP1β, NSP2, NSP3, NSP4, NSP7α, NSP7β, NSP8 and NSP9 may be included in the PRRSV RTC [4, 5, 46]. HSP70 may be recruited to enhance these NSPs stability and to assist their translocation into the RTC. Further studies are required to address the interactions of HSP70 with these NSPs, and to figure out how these interactions might regulate viral replication.