Zika Virus Infection of Sertoli Cells Alters Protein Expression Involved in Activated Immune and Antiviral Response Pathways, Carbohydrate Metabolism and Cardiovascular Disease

Zika virus (ZIKV), a re-emerging virus, causes congenital brain abnormalities and Guillain–Barré syndrome. It is mainly transmitted by Aedes mosquitoes, but infections are also linked to sexual transmissions. Infectious ZIKV has been isolated, and viral RNA has been detected in semen over a year after the onset of initial symptoms, but the mode of long-term persistence is not yet understood. ZIKV can proliferate in human Sertoli cells (HSerC) for several weeks in vitro, suggesting that it might be a reservoir for persistent ZIKV infection. This study determined proteomic changes in HSerC during ZIKV infections by TMT-mass spectrometry analysis. Levels of 4416 unique Sertoli cell proteins were significantly altered at 3, 5, and 7 days after ZIKV infection. The significantly altered proteins include enzymes, transcription regulators, transporters, kinases, peptidases, transmembrane receptors, cytokines, ion channels, and growth factors. Many of these proteins are involved in pathways associated with antiviral response, antigen presentation, and immune cell activation. Several immune response pathway proteins were significantly activated during infection, e.g., interferon signaling, T cell receptor signaling, IL-8 signaling, and Th1 signaling. The altered protein levels were linked to predicted activation of immune response in HSerC, which was predicted to suppress ZIKV infection. ZIKV infection also affected the levels of critical regulators of gluconeogenesis and glycolysis pathways such as phosphoglycerate mutase, phosphoglycerate kinase, and enolase. Interestingly, many significantly altered proteins were associated with cardiac hypertrophy, which may induce heart failure in infected patients. In summary, our research contributes to a better understanding of ZIKV replication dynamics and infection in Sertoli cells.


Introduction
Zika virus (ZIKV) belongs to the family Flaviviridae, and is a single-stranded positivesense RNA virus. Other members of the family include Japanese encephalitis virus (JEV), Yellow fever virus (YFV), West Nile virus (WNV) and Dengue virus (DENV). ZIKV was first discovered in Rhesus monkeys in Uganda's Zika rainforest in 1947 [1]. However, the virus remained undiagnosed for a long time due to the disease's nonspecific flu-like symptoms and a lack of diagnostic screening [2,3]. In 2007, ZIKV re-emerged in the Pacific islands, spreading to over 80 countries/territories, including Latin America, the United States, and Southeast Asia [4][5][6][7][8]. The virus has been linked to microcephaly

Protein Extraction and Quantification
The Sertoli cells infected with ZIKV and time-matched mock-treated cells were scraped from the culture plates after 3, 5 and 7 days of infection. Centrifugation at 600× g for 8 min pelleted the cells, which were then washed three times with sterile ice-cold PBS(Thermo Fisher Scientific, Waltham, CA, USA). The pelleted cells were then lysed in 4% SDS in 100 mM HEPES buffer pH 8.5 by sonication. Centrifugation at 14,000× g for 10 min at 4 • C was used to remove insoluble cellular components. Bradford Protein Assay was used to determine the protein concentrations in the supernatants (Bio-Rad, Hercules, CA, USA, Cat. 5000001).

Tandem Mass Tags (TMT) Mass Spectrometry Analyses and Protein Quantification
To determine the impact of ZIKV infection on the cellular proteome, a total of 18 protein samples were collected from ZIKV-and mock-infected Sertoli cells at 3, 5 and 7 days post-infection (dpi). Proteins were digested into peptides by the SP3 (single-pot solidphase-enhanced sample preparation) methods as described elsewhere [38,39]. In summary, peptides were eluted after digestion of the proteins with trypsin for 14 h at 37 • C. Sixplex TMT labeling was performed for mock and infected samples of the same time points following the manufacturer's (Thermo Fisher Scientific, Waltham, CA, USA) instructions. An equal amount of six TMT labeled samples were mixed together and 2D LC/MS/MS was performed using an Orbitrap Q Exactive HF-X instrument [40] (Thermo Fisher Scientific, Bremen, Germany). For identification of the peptide/proteins, ZIKV (Thai strain) and human (Uniprot 2016) databases were used as references. The intensities of TMT6 peptide level reporter tags were averaged over a ±0.1 Da window and corrected for isotopic overlap between channels using the batch-specific correction matrix provided. The sum of peptide level TMT6 reporter tag intensities for each protein was transformed into a log 2 scale for easier differential analysis.

Statistical and Bioinformatics Analyses
Initially, alterations in the levels of any individual protein expression were determined by the differences between Log 2 values (Delta log) of an infected and time-matched mock sample. Then, the delta Log 2 values were converted to fold-change for each of the proteins. The p-value was determined by the Students t-test (2 tails) and Z-score analyses based on the protein expression difference of all three replicates. p-value < 0.05 and Z-score values of ≥.96 σ and ≤−1.96 σ were considered significant as described before [41]. The lists of significantly altered proteins were uploaded into Ingenuity Pathway Analysis (IPA) software, and core analysis was done with a cut-off value p-value < 0.05 and fold change above 1.5 or below −1.5. The IPA core analysis predicted the top affected canonical pathways, bio-functions, interconnecting networks and upstream molecules based on these protein level changes. The Western blot image band intensities were quantified using Image J version1.53e software (NIH, Bethesda, MD, USA), and statistical analyses were performed by one-way or two-way ANOVA (p-values < 0.05) in GraphPad Prism version 6.0. MORPHEUS (Broad Institute, Cambridge, MA, USA), a free internet software program, was used to generate the heatmaps.

Infectivity of ZIKV and Its Cytopathic Effect in Primary HSerC
After infecting HSerC with ZIKV at MOI = 3, viral protein expression levels, which could result from increased expression, lower protein turnover, or a combination of both, and cytopathic effects were monitored at 3, 5 and 7 dpi, ZIKV infection did not induce any cytopathic effects ( Figure 1A). However, three viral proteins (NS1, NS3, and E) were expressed at all time points in the infected cells, with maximal levels at 3 dpi; by 5 and 7 dpi, their expressions had dropped significantly ( Figure 1B,C). Mass spectrometry analysis of Zika viral protein levels revealed that most of the proteins were at their highest levels at 3 dpi, with the exception of NS2B, NS4A, and C, which peaked at 5 dpi. All ZIKV proteins were expressed to the lowest level at 7 dpi ( Figure 1D). In our previous study, we did not observe any significant protein level alterations at the early stage (1 dpi) of replication. However, virus titer peaks at 5 dpi and declines at 7 dpi [36]. Therefore, we selected day 3 (mid), day 5 (peak viral titer) and day 7 (late stage) for subsequent proteomic analyses based on these observations.

The Impact of ZIKV Infection on the HSerC Cellular Proteome
TMT-based mass spectrometry was used to determine the proteomic alterations in protein levels caused by ZIKV infection in HSerC, which detected more than 6000 proteins from each sample ( Figure 2A). Among them, 4423 unique protein levels were significantly affected considering all three time points. A total of 2367 (853 up-regulated and 1514 downregulated), 2363 (1651 up-regulated and 712 down-regulated), and 1782 (1209 up-regulated and 573 down-regulated) proteins were significantly (p value < 0.05) affected at 3, 5 and 7 dpi, respectively (Table 1).    Heatmaps of the most affected proteins (Fold change ≥ 2.5 or ≤ −2.5; p-value < 0.05) revealed that certain protein levels were affected at specific time periods while other proteins were significantly affected at all times ( Figure 2B). The topmost up-regulated proteins were Regulator of G-protein signaling 5 Table 2). Principal component analysis (PCA) also showed that the protein samples were clustered by time point (Figure 2C).
The volcano plot analysis of the proteins revealed that the majority of the highly affected proteins were down-regulated at 3 dpi ( Figure 2D). In contrast, the most significantly up-regulated were at 5 and 7 dpi ( Figure 2E,F). The altered proteins belong to different classes of proteins, including enzymes, transcription regulators, kinases, transporters, peptides, cytokines, transmembrane receptors, phosphatases, G protein-coupled receptors, growth factors, ion channels, transcription regulators and others ( Figure 2G). We applied +/−1.5-fold fold-change cut-off with p < 0.05 for subsequent more complete bioinformatics analysis.

Impact of ZIKV Infection on Cellular Signaling Pathways and Function in HSerC
All proteins and their measured quantities at 3, 5 and 7 dpi were analyzed by Ingenuity Pathway Analysis (IPA) software to understand predicted impacts of ZIKV infection on cellular signaling pathways, bio-functions, and protein-protein networks. A total of 15, 31 and 13 Sertoli cell canonical pathways were predicted to be significantly affected at 3, 5 and 7 dpi, respectively, after ZIKV infection ( Figure 3A, Figure S1A). GP6 signaling pathway was inhibited at 3 dpi, but was activated significantly at 7 dpi. Natural killer cell signaling was significantly down-regulated only at 7 dpi. Seven signaling pathways were predicted to be significantly activated at all three-time points; they are hypercytokinemia/hyperchemokinemia in the pathogenesis of influenza, interferon signaling, Systemic Lupus Erythematosus in B cell signaling pathway, role of PKR in interferon induction and antiviral response, neuroinflammation signaling pathway, role of pattern recognition receptors in recognition of bacteria and viruses, and NAD signaling pathway (Figure 3, Figure S1A). Interestingly, a few canonical pathways were only affected at 5 dpi, e.g., IL-17 signaling, IL-6 signaling, Cardiac hypertrophy signaling (enhanced), HIF1α signaling, Glycolysis I, Gluconeogenesis I, PPAR signaling, HMGB1 signaling, Rac signaling, etc. IPA predicted the altered proteins were significantly (Z score >1.96 or <−1.96) associated with a total of 156 diseases and functions, which were divided into 55 for 3 dpi, 95 for 5 dpi and 63 for 7 dpi (Table S1 Pathways associated with infectious disease were predicted to be significantly inhibited, whereas those associated with antimicrobial response, inflammatory disease, neurological disease, tissue morphology, cell death and survival, and cellular movement were the major disease and cellular function categories predicted to be activated at all three time points ( Figure 3B; Figure S2). IPA also predicted a total of 801 unique upstream regulators to be activated/inhibited by ZIKV infection in Sertoli cells at 3 dpi (n = 400, activated, n = 279; inhibited 121), 5 dpi (n = 616, activated, n = 442; inhibited 174), and 7 dpi (n = 299, activated, n = 206; inhibited 93), which includes cytokines, enzymes, G-protein coupled receptors, growth factors, kinases, microRNAs, peptidases, phosphatase, transcription regulators, transmembrane receptor and transporters (Table S2). The top ten predicted upstream regulators are IFNG, IFNA2, TNF, IL1B, IRF7, IFNL1, Interferon-alpha, IFR1, IRGM and STAT1 ( Figure 3C). The functional analysis of the top 15 upstream regulators showed that they are associated with activation of T-lymphocytes, leukocytes, cells, recruitment of leukocytes, antiviral response, and RNA virus replication ( Figure S1B).
IPA also built protein-protein networks of the significantly affected proteins based on their direct or indirect interactions. There were 12, 9, and 10 protein-protein interaction networks (Score > 20, Molecules > 13) predicted by IPA analysis of the altered proteins at 3, 5 and 7 dpi, respectively. The predicted networks were associated with different cellular functions and diseases, including cell morphology, cellular assembly and organization, neurological disease, lipid metabolism, molecular transport, cardiovascular disease, connective tissue disorders, antimicrobial response, cell cycle, and endocrine system development (Table S3). Cancer, connective tissue disorders, organismal injury and abnormalities (Score 38, Focus Molecules 23) were the most affected predicted proteinprotein networks at 3 dpi, cardiovascular disease, cell death and survival, and connective tissue disorders (Score 42, Focus Molecules 25) were the most affected at 5 dpi, and antimicrobial response, and infectious diseases, inflammatory response (Score 41, Focus Molecules 22) were predicted to be the most affected at 7 dpi ( Figure 3D; Figure S2C,D).   Red and green represent up-regulation and down-regulation, respectively; gray proteins denote that they were recognized in the present study, but not significantly regulated; colorless proteins interact with molecules in the network, but were not identified in our study. Abbreviations. dpi = Days post infection.

HSerC Activates Immune Response against ZIKV Infection
To understand the impact of ZIKV infections across the three time points, we selected the list of 50 commonly affected proteins ( Figure 4A,B) and performed IPA core analysis. This analysis showed that the most affected proteins were predicted to be associated with canonical pathways involving replication of flavivirus, replication of RNA virus and in- terferon signaling pathways ( Figure 4C). The infectious disease, inflammatory response, neurological disease, and antimicrobial disease responses were the most affected disease and functions predicted by heatmap analysis of IPA ( Figure 4D). The protein-protein network analysis predicted their links with cell−to−cell signaling and interaction, infectious diseases, and post−translational modification ( Figure 4E).  The proteinprotein interaction networks affected by the commonly altered proteins. Red and green represent up-regulation and down-regulation, respectively; gray proteins denote that they were recognized in the present study, but not significantly regulated; colorless proteins interact with molecules in the network, but were not identified in our study. Abbreviations. dpi = Days post infection.
The immune response of antigen-presenting cells, immune response of phagocytes and leukocytes, activation of antiviral response, phagocytosis of cells and innate immune response also were predicted to be activated at 3, 5 and 7 days post-ZIKV infection ( Figure 5A). Several proteins involved in immune response regulating signaling pathways were also significantly stimulated by ZIKV infection, which includes interferon signaling, PKR interferon induction and antiviral response, role of pattern recognition receptor in recognition (PRRR) of bacteria and virus, Th1 pathway, HMGB1, TREM1, IL-17, IL-8, IL-6, IL-15 signaling, etc. ( Figure 5B). Across the three time points after ZIKV infection, interferon signaling was one of the most affected signaling pathways in Sertoli cells. ZIKV caused significant alteration of 12 protein levels that are predicted to regulate interferon signaling pathways ( Figure 5C). Based on the expression values of these proteins, IPA predicted a significant activation of the pathways at 3 dpi (Z-score 2.82), 5 dpi (Z-score 3.0), and 7 dpi (Z-score 3.0) ( Figure 5D). The immune response of antigen-presenting cells, immune response of phagocytes and leukocytes, activation of antiviral response, phagocytosis of cells and innate immune response also were predicted to be activated at 3, 5 and 7 days post-ZIKV infection ( Figure  5A). Several proteins involved in immune response regulating signaling pathways were also significantly stimulated by ZIKV infection, which includes interferon signaling, PKR interferon induction and antiviral response, role of pattern recognition receptor in recognition (PRRR) of bacteria and virus, Th1 pathway, HMGB1, TREM1, IL-17, IL-8, IL-6, IL-15 signaling, etc. ( Figure 5B). Across the three time points after ZIKV infection, interferon signaling was one of the most affected signaling pathways in Sertoli cells. ZIKV caused significant alteration of 12 protein levels that are predicted to regulate interferon signaling pathways ( Figure 5C). Based on the expression values of these proteins, IPA predicted a significant activation of the pathways at 3 dpi (Z-score 2.82), 5 dpi (Z-score 3.0), and 7 dpi (Z-score 3.0) ( Figure 5D).  (D) Activation of Interferon signaling pathway by ZIKV infection. Red and green represent upregulation and down-regulation, respectively; gray proteins denote that they were recognized in the present study, but not significantly regulated; colorless proteins interact with molecules in the network, but were not identified in our study. Abbreviations. dpi = Days post infection.
We identified several proteins associated with establishment of viral infection and replication that were significantly affected by ZIKV infection in HSerC ( Figure 6A). Based on their expressions, IPA predicted that proteins associated with Flaviviridae infection and replication were significantly inhibited ( Figure 6B). Interestingly, these analyses also predicted that proteins associated with replication of some other viruses such as Hepatitis C virus, Herpesviridae, murine herpesvirus 4, Orthomyxoviridae, and coronavirus were affected ( Figure 6B). These proteins associated with viral infection and replication are predicted to be found in the cytoplasm, nucleus, cell membrane, and extracellular space of Sertoli cells ( Figure 6C).
up-regulation and down-regulation, respectively. The number in each box shows the activation Zscore predicted by IPA. (C) Proteins in Interferon signaling pathways affected by ZIKV infection. (D) Activation of Interferon signaling pathway by ZIKV infection. Red and green represent up-regulation and down-regulation, respectively; gray proteins denote that they were recognized in the present study, but not significantly regulated; colorless proteins interact with molecules in the network, but were not identified in our study. Abbreviations. dpi = Days post infection.
We identified several proteins associated with establishment of viral infection and replication that were significantly affected by ZIKV infection in HSerC ( Figure 6A). Based on their expressions, IPA predicted that proteins associated with Flaviviridae infection and replication were significantly inhibited ( Figure 6B). Interestingly, these analyses also predicted that proteins associated with replication of some other viruses such as Hepatitis C virus, Herpesviridae, murine herpesvirus 4, Orthomyxoviridae, and coronavirus were affected ( Figure 6B). These proteins associated with viral infection and replication are predicted to be found in the cytoplasm, nucleus, cell membrane, and extracellular space of Sertoli cells ( Figure 6C).

ZIKV Infection Impacts Carbohydrate Metabolism in Sertoli Cells
At 5 dpi ZIKV caused significant activation of glycolysis (Z-score = 2.44) and gluconeogenesis (Z-score = 2.0) pathways ( Figure 3A). There were 18 Sertoli cell proteins associated with the glycolysis and gluconeogenesis pathway that were significantly affected by ZIKV infection. Eleven of their levels (SLC2A1, IL1B, PRP14, FABP1, ADCY10, PGK1, IL6, ENO2, ENO1, PGAM1 and SL39A14) were altered ( Figure 7A) at 5 dpi and predicted to significantly activate (Z score = 2.44) the glycolysis pathway ( Figure 7B). ZIKV infection significantly affected 20, 19 and 11 proteins related to carbohydrate metabolism and cellular energy production pathways at 3, 5 and 7 dpi, respectively ( Figure 7C; Figure S3). These proteins are associated with the synthesis of ATP and its concentration in cells, metabolism of carbohydrate, phosphatidic acid, D-glucose, D-hexose, monosaccharide, phospholipids and synthesis of carbohydrate and phospholipid.

ZIKV Infection Significantly Affects Proteins That May Increase Cardiovascular Disease
ZIKV infection significantly affected 20 proteins ( Figure 8D) involved in the cardia hypertrophy pathway and significantly activated (Z-score = 3.1) proteins within the path way at 5 dpi ( Figure 8A). However, at 3 dpi and 7 dpi, the proteins in this pathway wer

ZIKV Infection Significantly Affects Proteins That May Increase Cardiovascular Disease
ZIKV infection significantly affected 20 proteins ( Figure 8D) involved in the cardiac hypertrophy pathway and significantly activated (Z-score = 3.1) proteins within the pathway at 5 dpi ( Figure 8A). However, at 3 dpi and 7 dpi, the proteins in this pathway were not significantly impacted by ZIKV infection (Figure 8B,C). IPA has a database of 617 proteins associated with the increase of cardiovascular disease. Among these, 23, 28 and 14 proteins were significantly affected by 3, 5 and 7 days post-ZIKV infection, respectively. The significantly altered proteins are associated with atherosclerosis, ventricular dysfunction, myocardial dysfunction, cardiac lesion, hypertrophy of heart, morphology of heart and cardiovascular system and dysfunction of heart ( Figure 8E-G).

Validation of Mass Spec Data by Western Blot
Five proteins (CLIC1, SPARC, STAT1, STAT3 and PSMA2) were selected for valida tion based on their expression fold-change and availability of commercial antibodies Western blot was done ( Figure 9A), and the intensities of protein bands were measured to determine the fold difference between the infected and mock conditions. The fold change of each protein measured by Western blot at three different time points was com

Validation of Mass Spec Data by Western Blot
Five proteins (CLIC1, SPARC, STAT1, STAT3 and PSMA2) were selected for validation based on their expression fold-change and availability of commercial antibodies. Western blot was done ( Figure 9A), and the intensities of protein bands were measured to determine the fold difference between the infected and mock conditions. The fold change of each protein measured by Western blot at three different time points was compared side-by-side to the expression values determined by mass spectrometry ( Figure 9B). All five proteins followed the same trend of expression by Western blot as measured by TMT-mass spectrometry.

Discussion
The mechanism(s) underlining the long-term persistence of ZIKV in male semen is not yet clearly understood. The sexual transmission of the virus raises the risk of initiating outbreaks in non-endemic regions, even in the absence of a mosquito vector. Furthermore, the pathologic consequences of viral persistence in the male genital tract are unclear. In our previous study, to investigate ZIKV infection-induced HSerC proteome alterations, we applied SOMAscan, a multi-plexed targeted technology that can identify over 1300 proteins from each sample [36]. Although SOMAscan, as a targeted approach, can identify many non-abundant proteins, it is still limited in its detection limit to the specified 1305 proteins [42]. Therefore, in this study, we used a six-plex TMT-based mass spectrometry approach to determine additional HSerC proteome alterations to provide a broader insight on the impact of ZIKV infection. However, unlike the previous study, we excluded the 1 dpi time point due to a small number of significantly affected proteins [36] and in-

Discussion
The mechanism(s) underlining the long-term persistence of ZIKV in male semen is not yet clearly understood. The sexual transmission of the virus raises the risk of initiating outbreaks in non-endemic regions, even in the absence of a mosquito vector. Furthermore, the pathologic consequences of viral persistence in the male genital tract are unclear. In our previous study, to investigate ZIKV infection-induced HSerC proteome alterations, we applied SOMAscan, a multi-plexed targeted technology that can identify Viruses 2022, 14, 377 26 of 31 over 1300 proteins from each sample [36]. Although SOMAscan, as a targeted approach, can identify many non-abundant proteins, it is still limited in its detection limit to the specified 1305 proteins [42]. Therefore, in this study, we used a six-plex TMT-based mass spectrometry approach to determine additional HSerC proteome alterations to provide a broader insight on the impact of ZIKV infection. However, unlike the previous study, we excluded the 1 dpi time point due to a small number of significantly affected proteins [36] and included 7 dpi to analyze the impact of ZIKV on Sertoli cells at a later stage. In addition, as previously reported [36,37,43], Zika virus infection had no apparent cytopathic impact on Sertoli cells after infection ( Figure 1A). However, ZIKV replication [36] and viral protein expression ( Figure 1B,C) were significantly reduced at 7 dpi in Sertoli cells, but infectious virus has been detected for at least 6 weeks in these cells [37]. This indicates that ZIKV initially undergoes a robust infection in Sertoli cells, but it is subsequently controlled, likely by cellular immune responses and later it maintains a low level of persistence. In this study, we have seen that most of the significantly affected proteins were down−regulated at 3 dpi, and up−regulated at 5 dpi as observed before [36]. This confirms a consistent time−dependent switch of proteomic alteration caused by ZIKV infection in Sertoli cells. Among the most affected proteins, FBXO11 has been associated with Neurodevelopmental disorders, mental retardation, and autism [44,45]. However, KIF1A expression anomaly was linked with neurological disorders in children [46]. Therefore, the alteration of FBXO11 and KIF1A could be associated with microcephaly and Guillain-Barré syndrome that develop during and/or after ZIKV infection. UHRF2 is one of the central regulators of cell cycle machinery [47]. TYR is responsible for initiating the conversion of tyrosine to melanin, which is responsible for pigmentation of eye, skin and hair [48]. Abnormalities in TYR expression may cause albinism [49,50]. FOXQ1 is a transcription regulator that controls the cell cycle, cell proliferation and is associated with cancers [51][52][53][54], and that inhibits macrophage recruitment [55], and activates Wnt signaling [56].

HSerC Activates Immune Response against ZIKV Infection
The human testis maintains an immune-privileged environment, separated from the body cavity by the blood-testis barrier (BTB), made with Sertoli cells [57]. However, to combat invading microbial pathogens, the testis possesses a local immune defense system [28]. In response to ZIKV infection, HSerC induced changes in the levels of proteins predicted to activate a strong immune response at 3, 5 and 7 dpi. According to the analysis of the 50 predominantly affected proteins, the inflammatory and antimicrobial response pathways were significantly triggered after ZIKV infection of HSerC ( Figure 4D). The protein-protein interaction analysis also predicted antimicrobial response, infectious diseases, and inflammatory response as activated across the three-time points (Table S3). In addition, at least 17 cellular signaling pathways associated with immune response were predicted to be significantly activated in HSerC after ZIKV infection ( Figure 5B). In agreement with our findings, a previous study based on transcriptomic analysis [35] also determined that interferon signaling, IL-15, role of PKR interferon induction and antiviral response, HMGB1 and role of pattern recognition receptor in recognition of bacteria and virus were significantly affected by ZIKV infection of Sertoli cells. Further analysis of the significantly affected proteins ( Figure 5A) and upstream molecules ( Figure S1B) indicated that proteins involved in immune response of antigen-presenting cell pathways (APCs), phagocytes, leukocytes and T lymphocytes were significantly activated. Sertoli cells' potent immune response may have inhibited viral replication, as anticipated by IPA ( Figure 6B), also seen by the decreased quantity of viral proteins at 5 and 7 dpi ( Figure 1B,C). Interestingly, proteins associated with antiviral response against Flaviviridae may significantly suppress other human viruses such as Hepatitis C virus, Herpesvirus, and Coronavirus ( Figure 6A,B). This suggests that targeting commonly used host proteins or pathways might lead to the development of a broad-spectrum antiviral therapy in the future.

ZIKV Affects Proteins Involved in Carbohydrate Metabolism
Viruses use biomolecules from the host and induce anabolism to create the macromolecules needed for virion replication and assembly. As a result, it is no surprise that viral infection forces host cells to modify their metabolism in order to support effective virus replication [58]. Many oncogenic viruses use the glycolysis pathway, such as human papillomavirus (HPV), hepatitis C virus (HCV), hepatitis B virus (HBV), Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), and Adenovirus [59,60]. However, several non-oncogenic viruses were also found to activate the glycolysis pathways, including Herpes simplex virus 1 and 2 [61], and Human cytomegalovirus [58]. Dengue virus (DENV), another relative of ZIKV, depends on the glycolysis pathways for its replication [62]. High concentrations of glucose can restrict the growth of ZIKV in human kidney cells [63]. ZIKV infection increases glucose incorporation into the TCA cycle in mosquito cells, but the mechanism is not clear [64]. In HSerC, proteins involved in glycolysis and gluconeogenesis pathways were significantly activated by ZIKV infection (Figures 3A and 7B). In addition, the metabolism of D-fructose, monosaccharides, phosphatidic acid, phospholipids were also predicted to be significantly activated by the significantly altered proteins ( Figure 7C, Figure S3). Previously, we detected PGAM1, a key regulator of glycolysis and gluconeogenesis pathways, as significantly up-regulated by ZIKV infection in HSerC [36]. However, proteins involved in energy generation in Sertoli cells by lactate and lipid oxidization through the Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway [65] were significantly down-regulated by ZIKV infection (Figure 3A), as reported previously [36]. These results suggest that ZIKV hijacks carbohydrate metabolism to usurp cellular energy generation in HSerC. The glycolysis pathway could be a potential target against ZIKV and needs further investigation in the future.

ZIKV Infection Affects Proteins Associated with an Increase in Cardiovascular Disease
Surprisingly, we found many proteins involved in cardiac disease that were significantly affected ( Figure 8E-G) during ZIKV infection of HSerC. These proteins are associated with significant activation of atherosclerosis, ventricular dysfunction, myocardial dysfunction, and cardiac lesion ( Figure 8E-G). RGS5 is one of the topmost affected proteins detected in HSerC after ZIKV infection. It is a GTPase activator that protects against cardiac hypertrophy and cardiac fibrosis [66]. Cardiovascular disease was a frequently detected protein-protein network predicted by IPA at all three−time points after ZIKV infection (Table S3). In addition, levels of at least 20 proteins involved in the cardiac hypertrophy signaling pathway were significantly altered by ZIKV, causing significant predicted activation of the pathway (Figures 3A and 8A) at 5 dpi in HSerC. Thus far, cardiovascular complications have been associated with ZIKV infection by a case report [67], an observational study [68], and in an animal study [69]. Although identified by us in HSerC, the cardiac hypertrophy signaling pathway could be a possible mechanism of cardiac complications in ZIKV-infected patients. However, the current study has limitations, as it was performed in a non-cardiac cell line. To fully comprehend the link between ZIKV and cardiovascular abnormalities and its mechanism, extensive investigation is necessary.

Conclusions
Understanding the mechanisms of ZIKV persistence in the male genital tract is critical for treatment of the disease, antiviral/vaccine development and overall control of viral transmission. Multiple studies have suggested that Sertoli cells could be a potential reservoir for this viral persistence. Sertoli cells build the blood-testis barrier that maintains an isolated environment for optimal development of the germ cells and provides sufficient nutrients and signals for sequential differentiation into sperm by spermatogenesis [30]. ZIKV infection of Sertoli cells may impact sperm development and male fertility. Moreover, a clear understanding of the replication dynamics of the virus in an immune−privileged environment is necessary for antiviral or vaccine development. Previous studies of ZIKV−infected