Hsp90 inhibitors directly block HSV-1 DNA replication via inducing degradation of pUL42


 Background：Herpes simplex virus type I (HSV-1) is a member of the Alphaherpesvirinae family, which could initiate labial herpes caused by the reactivation of HSV-1 primary infection, and genital herpes caused by secondary infection, even causes herpes encephalitis. The purpose of this study is to study potential viral proteins and the mechanism of antiviral activity of new Hsp90 inhibitors (AT-533 and 17-AAG). In previous studies, the Hsp90 inhibitor geldanamycin has been confirmed to induce the abnormal localization of HSV-1 pUL30, but it is unclear whether Hsp90 inhibitors affect other viral replication proteins.Methods：The anti-HSV-1 activity of AT-533 and 17-AAG were evaluated by CCK-8 assay and fluorescent virus titer assay. The mechanism of action against HSV-1 DNA replication was determined by the previous study and this research. Then we assayed the viral RNA transcription, viral DNA replication and protein expression, respectively. In addition, we identified the replication-related proteins that interact with Hsp90 through protein docking and co-immunoprecipitation, and determined the effects of AT-533 and 17-AAG on the expression of replication-related proteins.Results：CCK-8 and fluorescent virus titer determination showed that Hsp90 inhibitors hasve good anti-HSV-1 activity on HFF cells. Interestingly, AT-533 significantly reduces the synthesis of viral DNA, but it does not affect the expression of viral mRNA. Further, protein docking stimulation and co-IP assay revealed that DNA polymerase pUL42 interacted with Hsp90, which was largely degraded via autophagy by Hsp90 inhibitors. However, AT-533 does not affect the expression of other replication-related proteins, such as ssDNA binding protein UL9 and helicase-primerase UL5/8/52. The results suggested that Hsp90 inhibitors directly inhibited viral DNA replication by affecting the stability of DNA polymerase pUL42.Conclusions：Our results indicate that Hsp90 inhibitors may be potential candidates for HSV-1 therapy by directly inhibiting DNA replication, especially for ACV resistant strains.


Introduction
Herpes simplex virus-1 (HSV-1), a common human pathogenic viruse, belongs to α herpesvirinae family [1]. HSV-1 could initiate labial herpes caused by the reactivation of HSV-1 primary infection, and genital herpes caused by secondary infection even causes herpes encephalitis [2]. Speci cally, HSV-1 contains at least 7 conserved protein to effect DNA replication, such as origin-binding protein UL9, singlestranded DNA binding protein ICP8, DNA polymerase complex UL30/42 and helicase-primase complex UL5/8/52 [3]. Among the most important HSV-1 proteins, HSV-1 DNA polymerase and the early enzyme thymidine kinase (TK) play a key role in the virus replication process, so drugs targeting these proteins may disrupt the virus replication cycle [4] . At present, nucleic acid analogs have been used as anti-HSV-1 drugs in the clinical frontline, which target the stage of viral DNA replication, especially acting on thymidine kinase (TK). The representative drug is acyclovir and its derivatives. Due to the limitations of nucleoside analog drugs, patients who take these drugs for a long time to treat HSV-1 infection may induce drug-resistant virus strains and adverse reactions [5].
Recently, many studies have indicated that heat shock protein 90 (Hsp90) is a promising broad-spectrum antiviral host factor, which is required by many viruses for multiple phases of their life cycle including viral entry, nuclear import, transcription, and replication [6]. Previous research indicated that Hsp90 inhibitor geldanamycin reduced viral titers and induced the abnormal location of HSV-1 DNA polymerase catalytic subunit pU L 30 [7]. However, the particular interaction between Hsp90 and HSV-1 replicationrelated proteins still unknown. In this study, we further studied the potency and mechanism of antiviral activity of novel Hsp90 inhibitor AT-533 against HSV-1 DNA replication-related proteins and ACV-resistant strains.
Brie y, HFF cells were cultured in 96-wells plates and reached at 85% cell con uence. Various concentrations of compound were added to the plate, with each concentration having three replicates.
After 48 h of incubation, 10 µL CCK-8 solution (5 mg/mL) was added to each well, and the plate was incubated for 1h protected form light. Then, the plates incubated for 10 min at room temperature with gently shaking. The optical density (OD) at 450 nm was measured for each well with an enzyme-labeled reader (Bio-Rad, Hercules, CA, USA). The 50% cytotoxicity concentration (CC 50 ) was de ned as the concentration to reduce 50% cell viability.

Viral titer determination
Vero cells were cultured in 96-wells plates. And the next day, Vero cell monolayers were incubated with eleven-fold serial dilutions of 100 µl HSV-1 treatment and incubated at 37°C and 5% CO2 for 72h. Morphological changes of the cells (cytotoxic effects, CPEs) were observed every day under inverted microscope and cell viability was calculated based on CPEs. Control were cells treated only with maintenance medium DMEM. The plaque assay was carried out in triplicate. Virus was quanti ed by serial dilution and titration assay. The TCID 50 (50% tissue culture infectious dose) was calculated using the formula of Reed and Muench method Log 10 50% end point dilutions = log 10 of dilution showing a mortality next above 50% -(difference of logarithms × logarithm of dilution factor). Difference of logarithms = [(mortality at dilution next above 50%)-50%] / [(mortality next above 50%) -(mortality next below 50%)] [9].
Antiviral effect of GFP-HSV-1 HFF cells were cultured in 96-wells plates, and the second day cell monolayers were incubated with eleven-fold serial dilutions of 100 µl Hsp90 inhibitor treatment and infected MOI = 1 GFP-HSV-1 at 37°C and 5% CO2 for 72h [10]. The plaque assay was carried out in triplicate. GFP uorescence was quanti ed using a Synergy Neo HTS Multi-Mode Reader (Biotek Instruments). The relative uorescence intensity was calculated using the formula as follows: Relative uorescence intensity= (Hsp90 inhibitors treatment uorescence intensity) / (GFP-HSV-1 control uorescence intensity)×100%.
CPEs Cell viability assay HFF cells were cultures in 96-well plates. And the next day, HFF cell monolayers were incubated with eleven-fold serial dilutions of 100 µL HSV-1 treated with or without Hsp90 inhibitors at 37°C and 5% CO 2 .
After 72h, 10 µL CCK-8 solution was added to each well, and incubated for 1h in dark. Then, the plates incubated for 10 min at room temperature with gently shaking. The optical density (OD) at 450 nm was measured for each well with an enzyme-labeled reader (Bio-Rad, Hercules, CA, USA). The CPEs inhibition ratio was calculated by the formula as follows. The CPEs inhibition ratio= [(Absorption of treatment groups) -(Absorption of cell control)] / [(Absorption of virus control) -(Absorption of cell control)] ×100% [11].
The detection of DNA synthesis HFF cells were cultured in 12-well plates. And the second day cells infected with HSV-1 (MOI = 20) were incubated with or without AT-533 or 17-AAG for 12 h. Viral DNA was extracted using GeneJET Viral DNA and RNA Puri cation Kit (Thermo). Real-time PCR assay was used to quantify the viral DNA. Then the HSV-1 DNA copy numbers were expressed relative to the virus control groups. The primer pairs are seen at Additional le 1: Table S2.

Real-time PCR (Q-PCR)
HFF cells were cultured in 12-well plates. And the second day cells infected with HSV-1 (MOI = 20) were incubated with or without AT-533 or 17-AAG for 2, 3, 4 h, respectively. Total RNA was isolated using Trizol (Invitrogen) and subjected to cDNA synthesis using a PrimeScript RT reagent kit (Takara). Real-time PCR (RT-PCR) was conducted to determine the expression levels of UL9, UL30, UL42 of HSV-1/F and at 2, 3 and 4h.p.i, respectively. The primer pairs were the same as described above.
Protein docking stimulation ZDock (http://zdock.umassmed.edu/) was manipulated to predict the structures of protein-protein interaction and symmetric multimers. Prediction of protein-protein interaction was in three steps as follows [12] . Firstly input ligand and receptor protein structures and choose the ZDock version options; Secondly select the blocking/contacting residues; Thirdly view docking results online or download in Pymol. Binding a nity (∆G) and dissociation constant (K d ) predicted values for the protein-protein interactions was obtained from docking results.

Molecular docking stimulation
LeDock (http://www.lephar.com/index.htm) was manipulated to stimulate the docking between small molecular compounds and proteins. Docking binding a nity (∆G) 0 kJ/mol indicated ligand molecules can spontaneously bind to receptor proteins, while (∆G) 5 kJ/mol indicated both of them can bind stably [13]. The target proteins in all networks were obtained 3D structures from the RCSB PDB database (http://www.rcsb.org/) [14]. 2D structures of all compounds was obtained from ZINC (http://zinc.docking.org/) [15]. Top10 docking complexes were docked between each protein and ligand, and chosen the complexes with the smallest binding energy.
Co-immunoprecipitation (co-IP) HFF cells were infected with HSV-1 for 12h in 100 mm 2 ask dish, which were collected and lysed in 100 µL SDS Lysis Buffer (Beyotime, China) containing 1% PMSF and centrifuged at 12,000 g. The supernatant was divided into two parts, one for input and other was incubated with IgG (normal mouse or rabbit IgG, primary antibody) at 4℃ overnight. Then the mixture was treated with 35µL of volume of Protein A/G magnetic beads at 4℃for 2 h. Next, the immune-precipitates were collected, washed three time with PBS, and re-suspended in 30mL 1*SDS-PAGE buffer (Beyotime, China). Finally, the samples were boiled for 10 min and analysed by western blotting.
Plasmid transfection HEK293T cells seed in 12-well plates, and the second day the mentioned plasmids were performed with Lipofectamin-3000 transfection reagent according to the manufacturer's instructions (Invitrogen). Brie y, 500ng of the corresponding plasmids and Lipofectamine 3000 reagent were diluted in 100µL Opti-MEM I reduced serum medium (Invitrogen) and the diluted plasmids were then added to the Lipofectamine 3000 (1:1 ratio), mixed, and incubated at room temperature for 10 min. The transfection mixture was then added to cells at 60-70%, and was replaced with esh 2% FBS DMEM after transfection 6h.
Western blotting HFF cells were seeded in 12-well plates with the density of 1.5 × 10 6 cells/ well. Until 85% cell con uence, cells were infected with HSV-1 (MOI = 20) at 37°C for 4 h. Therefore, DMEM maintenance medium with or without AT-533 or 17-AAG was added. At 12h post infection, the cells were washed three times with PBS, and were lysed with 1*SDS-PAGE buffer (Beyotime). The equal amount (40 µg/sample) proteins were subjected to Western Blot analysis.
Immuno uorescence assay HFF cells were cultured in 12-wells plates, and second day cells infected with HSV-1 (MOI = 20) at 37°C for 4h for viral DNA replication. Cells were transferred into maintain medium with or without AT-533 or 17-AAG and incubated for 8h. Cells were xed for 15 min with 4% paraformaldehyde (PFA) and permeabilized with 0.01% Triton X-100 for 5min, and subsequently incubated with anti-UL42 antibody (Abcam) at 4℃ overnight and Alexa Fluor 488 (1:1000) secondary antibody (Invitrogen) at room temperature for 1h. After each step the slides were washed triplicate with PBS, and which nally were preserved with PBS. Finally, the nuclear staining with 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes) was performed for 15min. Fluorescence was recorded in a uorescence microscopy (Canon).

Statistical analysis
Research data were calculated as the mean ± SD, and statistical signi cance were determined by the student's t test. The statistical signi cance were determined by P values (P < 0.05). More than three times was performed at each time.

Results
Cytotoxicity and anti-HSV-1 activity of AT-533 and 17-AAG To examine the cytotoxic effect of two potent Hsp90 inhibitors AT-533 and 17-AAG on HFF cells, CCK-8 assay was used. Different concentrations of Hsp90 inhibitors were used. AT-533 exhibited a larger cytotoxicity than 17-AAG (Fig. 1A), and achieved TC 50 (50% toxicity concentrations) of AT-533 and 17-AAG (Table 1). Hence, to examine the antiviral activities of these Hsp90 inhibitors, CPE (virus-induced cytopathic effect) reduction assay was used [11,16,17]. AT-533 (0.125 µM) and 17-AAG (0.20 µM) exhibited reasonable inhibition of induced CPE (Fig. 1B). To further clarify the antiviral effect of Hsp90 inhibitors, obtaining IC 50 (50% inhibition concentrations) in Table 1,antiviral assay of GFP-HSV-1 was used (Fig. 1C) [10,18]. Morphologically, gradient Hsp90 inhibitors were exhibited well recovery of HSV-1 cytopathic effects with concentration dependent (Fig. 1D). We next investigated anti-GFP-HSV-1 e cacy of HFF cell. GFP-HSV-1 was used to infect cells and the Hsp90 inhibitors were treated at the same time. Infected cells were imaged using a uorescence microscope at 12h.p.i, and the treatment of Hsp90 inhibitors had preferable uorescence inhibition with concentration dependent (Fig. 1E). Regulation of HSV-1 replication phase Next, we analyzed regulation of HSV-1 replication phase was affected by Hsp90 inhibitors AT-533 and 17-AAG.The detection of DNA synthesis was performed to demonstrate that AT-533 and 17-AAG signi cantly reduced genome copy production ( Fig. 2A).To evaluate whether Hsp90 exerts speci c function in HSV-1 immediately early and early life cycle, AT-533 and 17-AAG were added at 1h.p.i and obtained at different time points (2 ~ 6h.p.i) in HSV-1-infected HFF cells (Fig. 2B).Cell monolayers were then xed and extracted DNA, both inhibitors signi cantly reduced the HSV-1 DNA production at different time points(2 6h.p.i). Notably, HSV-1 that was not treated with Hsp90 inhibitors started to replicate in large numbers at 4h.p.i (Fig. 2C). Besides, to analyze the effects of Hsp90 inhibitors on HSV-1 gene expression, the mRNA expression levels of viral immediate-early gene (α0) were quanti ed at 3 ~ 6h.p.i, respectively (Fig. 2D). Both AT-533 and 17-AAG treatment signi cantly reduced the mRNA expression of viral immediate-early gene (α0). To further clari ed whether Hsp90 exerts directly or indirectly function in HSV-1 replication phase, AT-533 and 17-AAG were added at different time points(2,3,4h.p.i) and obtained at 12h.p.i (Fig. 2D). Hsp90 inhibitors treatment both signi cantly reduced the DNA copy number of UL9,UL30 and UL42 (Fig. 2D).Interestingly, Hsp90 inhibitors treatment could not reduce the mRNA expression of viral gene UL9,UL30 and UL42 at 4h.p.i and signi cantly reduced at 2,3h.p.i Generally, HSV-1 treated in HFF cells largely initiated DNA replication at 4h.p.i, meanwhile Hsp90 inhibitors treatment directly reduced DNA copy numbers instead in uencing viral gene transcriptions.
A.HFF cells was infected with HSV-1(MOI = 1) and extracted viral DNA to assess infectious and progeny virus DNA copy number at 12, 24, 48, 72h.p.i B. Diagrammatic sketch of time-addition assay to assess HSV-1 DNA copy number and mRNA expression. C.HSV-1 DNA copy number with or without AT-533 and 17-AAG was detected at different time points (2,3,4,5h.p.i). D.HSV-1 mRNA expression was detected at different time points (2,3,4,5h.p.i). E.Diagrammatic sketch of HFF cells treated with Hsp90 inhibitors in different time points to assess HSV-1 DNA copy number and mRNA expression. F.HFF cells was infected with HSV-1(MOI = 20) and treated with Hsp90 inhibitors at different time points (2,3,4h.p.i), and then viral DNA copy number was detected. G. HFF cells was infected with HSV-1(MOI = 20) and treated with Hsp90 inhibitors at different time points (2,3,4h.p.i), and then viral mRNA expression was detected.

Interaction between Hsp90 and HSV-1 replication-related proteins
In previous researches, Hsp90 inhibitor geldanamycin induced the abnormal location of HSV-1 DNA polymerase catalytic submit UL30, however few direct experimental evidences proved the interaction between Hsp90 and UL30 or other replication-related proteins [7].Thus,we further investigated the interaction and function between Hsp90 and HSV-1 replication-related protein.To prepare for the protein modeling and construct the viral proteins model, phylogenetic analysis of HSV-1 DNA polymerase UL30 and UL42 were individually retrieved from GenBank and showed in Fig. 3A&B. The crystal structure of DNA polymerase catalytic subunit UL30 (PDB:2GV9) and processivity factor UL42 model from Swissmodel (PDB:1DML) interacted with Hsp90 (PDB:4BQG) were protein docking stimulated (Fig. 3C&D).Hence, Homology analysis of HSV-1 U L 30/42 and other Herpesviridae DNA polymerase were exhibited (Table 2&3). Altogether, the binding a nity (∆G), dissociation constant (K d ) (Table 4) and higher number of protein-protein contacts (Table 5) between Hsp90 and viral proteins were showed. In conclusion, protein docking stimulation were proved that Hsp90 interact with both UL30 and UL42.To further con rm the interaction between Hsp90 and HSV-1 DNA polymerase, co-IP assay were manipulated (Fig. 3E).HFF infected with HSV-1 treatment with DMSO were indicated Hsp90 interact with UL42, and at the same time treated with 17-AAG were indicated the reducing interaction between Hsp90 and UL42.In short,Hsp90 interacted with HSV-1 DNA polymerase UL42 via protein docking stimulation and CO-IP assay.Because of the insu ciency of commercial HSV-1 UL30 antibody,the interaction between Hsp90 and UL30 is still need further studies.    Fig. 4A. Hsp90 inhibitors treatment reduced the protein expression of viral early proteins UL42 and host protein AKT instead of in uencing the protein expression of Hsp90 and ICP8, which indicated Hsp90 inhibitors blocked the activity of Hsp90 and reduced the client protein expression of UL42 instead of ICP8. Interestingly, Hsp90 inhibitors reduced the protein expression of UL42 only in early stage(2,4h.p.i) rather than 6,8h.p.i, which was consistent with time-addition DNA and mRNA detection. To further con rm the key time point of Hsp90 affecting DNA replication, HFF cells synchronously treated with HSV-1(MOI = 20) and Hsp90 inhibitors at 4h.p.i as showed in Fig. 4B, AT-533 and 17-AAG didn't affect the protein expression of ICP8 and affected UL42 indeed. To prepare plasmids transfection in HEK293T cells, the cytotoxicity detection of AT-533 and 17-AAG in HEK293T were assayed (Fig. 4B).To prepare the plasmid transfection in HEK293T cells, the CCK8 assay of AT-533 and 17-AAG in HEK293T cells are used. AT-533 exhibited a larger cytotoxicity than 17-AAG which was showed in Fig. 4C. HEK293T cells were transfected with HA-UL42 and HA-Vector plasmid, and at 6 h.p.i ash 2% DMEM were added with and without AT-533 or 17-AAG.AT-533 and 17-AAG treatment reduced the HA-UL42 expression and didn't affect the transfection of HA-Vector (Fig .4D).Consistently, the results of immuno uorescence assay was the same as western blotting of HA-UL42 transfection treated with Hsp90 inhibitors (Fig. 4E).Verifying the functionality and importance of HA-UL42 plasmid in HEK293T cells, which were rst infected with HSV-1 (MOI = 20)with or without Hsp90 inhibitors,and transfected with HA-UL42 to detect DNA production (Fig. 4F).Over-expression of HA-UL42 in HSV-1 infection with AT-533 and 17-AAG could restore the Hsp90 inhibitors inducing DNA replication reduction. In short, AT-533 and 17-AAG via suppressing viral and plasmid UL42 expression hindered HSV-1 DNA replication.
Hsp90 inhibition induced autophagy-mediated degradation of UL42 Next, we attempted to identify the speci c mechanisms responsible for the Hsp90 inhibition-induced UL42 degradation. In our previous study and those of others, Hsp90 client proteins were degraded via two main ways such as ubiquitination-proteasome pathway and autophagy-induced pathway. Hence, we rst assessed the levels of ubiquitinated proteins in HFF cells treated with different Hsp90 inhibitors and found that all treatments did not accumulate the polyubiquitin proteins (Fig. 5A). Besides, the proteasome inhibitors MG-132 was treated and did not restore the UL42 reduction of Hsp90 inhibitors inducing. Consistently, HEK293T cells also were treated with or without Hsp90 inhibitors and MG-132, which was not restored the reduction of HA-UL42 (Fig. 5B). To examined whether Hsp90 inhibition-induced UL42 degradation was dependent on the autophagy pathway. HFF cells were infected with HSV-1 (MOI = 20) and treated with or without concentration gradient Hsp90 inhibitors and Chloroquine (CQ), an inhibitor of the fusion of the lysosome and autophagosome. Chloroquine (CQ) could reverse the degradation of UL42 mediated by Hsp90 inhibition (Fig. 5C).Similarly,HEK293 cells were transfected with HA-UL42 and treated with Hsp90 inhibitors and CQ, as a result CQ treatment restored the degradation of UL42 (Fig. 5D).
Effects of AT-533 and 17-AAG on helicase-primase UL5/8/52 HSV-1 helicase-primase UL5/8/52 have the function of untying DNA double strands and coordinating the synthesis of leading and lagging chains. It is also indispensable for DNA proper and e cient synthesis. Therefore, to further examine the effect of AT-533 and 17-AAG on helicase-primase UL5/8/52, the immuno uorescence assay was used. HEK293T cells were transfected with Flag-UL5 and treated with or with AT-533 and 17-AAG after 6 hours post transfection. Hsp90 inhibitors didn't affect the protein expression of UL5 as showed in Fig. 6A. Consistently, Hsp90 inhibitors treatment were not reduced the protein expression of HA-UL8 and EGFP-UL52 (Fig. 6B&C).
Effects of AT-533 and 17-AAG on ACV-resistant HSV-1 Finally, we evaluated the potent antiviral activity of AT-533 and 17-AAG on ACV-resistant HSV-1 via performing viral titer determination and DNA synthesis detection. Vero cells were infected with HSV-1/153 (MOI = 1) and Hsp90 inhibitors, after 24h.p.i were extracted to analysis viral titer (Fig. 7A). Indeed, AT-533 and 17-AAG treatment reduced the viral titer of HSV-1/153 with favorable concentration dependent.
Therefore, the extraction from above was manipulated for viral DNA detection (Fig. 7B). In addition, AT-533 and 17-AAG reduced the DNA production of HSV-1/153.Consistently, AT-533 and 17-AAG treatment reduced the viral titer and DNA production of HSV-1/Blue (Fig. 7C&D). Among these data, Hsp90 inhibitors induced degradation of UL42 may played critical roles in antiviral activities of ACV-resistant HSV-1.

Discussion
Several studies have demonstrated that Hsp90 participates in many HSV-1 infectious stages including early and late stages, which indicated Hsp90 is a promising candidate of novel drug targets [19]. In our laboratory, previous studies found that Hsp90 could promote capsid protein nuclear transport via interacting with acetylated α-tubulin [20]. Further, Hsp90α knockdown or inhibition treated with 17-AAG and SNX-2112 signi cantly inhibited the promoter activity of HSV-1 α genes via destroying the stability of VP16 [21]. In addition, novel Hsp90 inhibitor AT-533 could hinder HSV-1 nuclear egress and assembly [22].
Many studies have described the function of Hsp90 in HSV-1 DNA replication stage. For example, Hsp90 inhibitor geldanamycin reduced viral titers and induced the abnormal location of HSV-1 DNA polymerase catalytic subunit pU L 30 [7,23]. However, the function of Hsp90 inhibitors and the interaction of Hsp90 between other HSV-1 replication-related proteins is still unknown.
In particular, it has been reported that Hsp90 has a regulatory roles in different virus particularly in DNA replication stages including DNA viruses, negative chain RNA virus and double chain RNA viruses [24].
ORF29F, is a single-stranded DNA binding protein in Varicella-zoster virus (VZV), whose nucleic localization is depend in the activity of Hsp90 [25]. Nuclear transport of epstein-barr virus (EBV) DNA polymerase catalytic subunit BALF5 is dependent on the molecular chaperone Hsp90 [26].Hence, the formation of dengue virus DNA polymerase NS5 protein was required by Hsp90 [27]. These ndings indicated that Hsp90 likely plays important roles in viral replication. In previous research, Hsp90 inhibitors geldanamycin induced the abnormal location of HSV-1 DNA polymerase catalytic subunit pU L 30 indeed.
However, explicit and direct research data of interaction between Hsp90 and UL30 is absent. Hence, in this study we focused on elucidating the role and speci c mechanism of Hsp90 inhibitors in DNA replication phase, and assessing the antiviral activity especially in ACV-resistant strains. The abuse of acyclovir and its derivatives caused the gene mutation of HSV-1 Thymokinase (TK) and DNA polymerase UL30, as a consequence ACV-resistant strains was bloomed dramatically [29,30].
Notably, Hsp90 inhibitors AT-533 and 17-AAG reduced the viral titer and infectious DNA production of ACV-resistant strains (HSV-1/106 and HSV-1/Blue). The DNA polymerase processivity factor UL42 may play a indispensable roles in antiviral effect upon ACV-resistant strains, which is still need to further explore and research.

Conclusion
Summarizing these data, novel Hsp90 inhibitor AT-533 is assessed to be a potential candidate at HSV-1 replication phase, especially for ACV-resistant strains. First of all, the time-addition detection of DNA copy number and mRNA expression indicated Hsp90 inhibitors AT-533 and 17-AAG directly block HSV-1 DNA replication phase. Secondly, protein docking stimulation and co-immunoprecipitation approved pUL42 interacted with Hsp90 as its client proteins. Besides, the results of western blot indicated Hsp90 inhibitors AT-533 and 17-AAG induced the degradation of pUL42 via autophagy pathway. Finally, Hsp90 inhibitors AT-533 and 17-AAG exhibited favorable antiviral activity towards ACV-assistant strains, which indicated pUL42 may be an antiviral active target for ACV-assistant strains. Comparing with previous work, this study identi ed pUL42 as a Hsp90 client protein in the HSV-1 replication stage and potential antiviral active target for ACV-assistant strains. It laid a solid theoretical foundation for other scholars to develop new anti-virus strategies and agents by focusing pUL42 as anti-virus targets.

Declarations
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