PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2.

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations.

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Chemical inactivation strategies for SARS-CoV-2-infected cells and organoids.

Infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, conducted in high-containment laboratories, requires transferring samples to lower containment labs for downstream applications, mandating sample inactivation. Here, we present a stepwise protocol for chemical inactivation of SARS-CoV-2 virus in culture supernatants or within infected cells and organoids, using eight chemical reagents validated via plaque assays. Additionally, we describe steps for troubleshooting virus inactivation, titer calculation, and log reduction. This protocol offers valuable resources for the COVID-19 research community, providing essential tools to advance research on this virus.

BoLA-DRB3 Polymorphism Associated with Bovine Leukemia Virus Infection and Proviral Load in Holstein Cattle in Egypt.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, the most prevalent neoplastic disease of cattle worldwide. The immune response to BLV and disease susceptibility and resistance in cattle are strongly correlated with the bovine leukocyte antigen (BoLA)-DRB3 allelic polymorphism. BLV infection continues to spread in Egypt, in part because the relationships between BLV infection, proviral load in Egypt, and BoLA-DRB3 polymorphism are unknown. Here, we identified 18 previously reported alleles in 121 Holstein cows using a polymerase chain reaction sequence-based typing method. Furthermore, BoLA-DRB3 gene polymorphisms in these animals were investigated for their influence on viral infection. BoLA-DRB3*015:01 and BoLA-DRB3*010:01 were identified as susceptible and resistant alleles, respectively, for BLV infection in the tested Holsteins. In addition, BoLA-DRB3*012:01 was associated with low PVL in previous reports but high PVL in Holstein cattle in Egypt. This study is the first to demonstrate that the BoLA-DRB3 polymorphism confers resistance and susceptibility to PVL and infections of BLV in Holstein cattle in Egypt. Our results can be useful for the disease control and eradication of BLV through genetic selection.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Preparing for the next viral threat with broad-spectrum antivirals.

There is a large global unmet need for the development of countermeasures to combat hundreds of viruses known to cause human disease and for the establishment of a therapeutic portfolio for future pandemic preparedness. Most approved antiviral therapeutics target proteins encoded by a single virus, providing a narrow spectrum of coverage. This, combined with the slow pace and high cost of drug development, limits the scalability of this direct-acting antiviral (DAA) approach. Here, we summarize progress and challenges in the development of broad-spectrum antivirals that target either viral elements (proteins, genome structures, and lipid envelopes) or cellular proviral factors co-opted by multiple viruses via newly discovered compounds or repurposing of approved drugs. These strategies offer new means for developing therapeutics against both existing and emerging viral threats that complement DAAs.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Comparison of the inactivation capacity of various UV wavelengths on SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a worldwide pandemic. Ultraviolet (UV) is regarded as a very powerful tool against SARS-CoV-2. However, the inactivating effects of different UV wavelengths on SARS-CoV-2 under the same conditions have hardly been compared. Here, we showed that SARS-CoV-2 cultured in Dulbecco's modified Eagle's medium and 2% fetal bovine serum was efficiently inactivated by irradiation with 222, 254, and 265 wavelengths UV, but not at 308 nm. In addition, it was revealed that UV absorption by DMEM-2% FBS is very efficient at 222 nm. Our results present potentially important information for selecting the optimum UV wavelength according to the application.

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for antiviral strategies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

Association between BoLA-DRB3 polymorphism and bovine leukemia virus proviral load in Vietnamese Holstein Friesian cattle.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. Polymorphism in bovine leukocyte antigen (BoLA)-DRB3 allele can influence the host immune response to pathogens, including BLV. However, association between specific BoLA-DRB3 alleles and BLV proviral load (PVL), which is a useful index for estimating disease progression and transmission risk, in Vietnamese cattle are unknown. Here, association study of BoLA-DRB3 allele frequency between cattle with high or low PVL demonstrated BoLA-DRB3*12:01 associates with high PVL in Vietnamese Holstein Friesian (HF) crossbred cattle. This is the first study to demonstrate that BoLA-DRB3 polymorphism confers susceptibility to BLV high PVL in HF crossbred kept in Vietnam. Our results may be useful in disease control and eradiation for BLV through genetic selection.

UVC disinfects SARS-CoV-2 by induction of viral genome damage without apparent effects on viral morphology and proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a pandemic threat worldwide and causes severe health and economic burdens. Contaminated environments, such as personal items and room surfaces, are considered to have virus transmission potential. Ultraviolet C (UVC) light has demonstrated germicidal ability and removes environmental contamination. UVC has inactivated SARS-CoV-2; however, the underlying mechanisms are not clear. It was confirmed here that UVC 253.7 nm, with a dose of 500 µW/cm2, completely inactivated SARS-CoV-2 in a time-dependent manner and reduced virus infectivity by 10-4.9-fold within 30 s. Immunoblotting analysis for viral spike and nucleocapsid proteins showed that UVC treatment did not damage viral proteins. The viral particle morphology remained intact even when the virus completely lost infectivity after UVC irradiation, as observed by transmission electronic microscopy. In contrast, UVC irradiation-induced genome damage was identified using the newly developed long reverse-transcription quantitative-polymerase chain reaction (RT-qPCR) assay, but not conventional RT-qPCR. The six developed long RT-PCR assays that covered the full-length viral genome clearly indicated a negative correlation between virus infectivity and UVC irradiation-induced genome damage (R2 ranging from 0.75 to 0.96). Altogether, these results provide evidence that UVC inactivates SARS-CoV-2 through the induction of viral genome damage.

SARS-CoV-2 Disinfection of Air and Surface Contamination by TiO2 Photocatalyst-Mediated Damage to Viral Morphology, RNA, and Protein.

SARS-CoV-2 is the causative agent of COVID-19, which is a global pandemic. SARS-CoV-2 is transmitted rapidly via contaminated surfaces and aerosols, emphasizing the importance of environmental disinfection to block the spread of virus. Ultraviolet C radiation and chemical compounds are effective for SARS-CoV-2 disinfection, but can only be applied in the absence of humans due to their toxicities. Therefore, development of disinfectants that can be applied in working spaces without evacuating people is needed. Here we showed that TiO2-mediated photocatalytic reaction inactivates SARS-CoV-2 in a time-dependent manner and decreases its infectivity by 99.9% after 20 min and 120 min of treatment in aerosol and liquid, respectively. The mechanistic effects of TiO2 photocatalyst on SARS-CoV-2 virion included decreased total observed virion count, increased virion size, and reduced particle surface spike structure, as determined by transmission electron microscopy. Damage to viral proteins and genome was further confirmed by western blotting and RT-qPCR, respectively. The multi-antiviral effects of TiO2-mediated photocatalytic reaction implies universal disinfection potential for different infectious agents. Notably, TiO2 has no adverse effects on human health, and therefore, TiO2-induced photocatalytic reaction is suitable for disinfection of SARS-CoV-2 and other emerging infectious disease-causing agents in human habitation.

Bovine major histocompatibility complex (BoLA) heterozygote advantage against the outcome of bovine leukemia virus infection.

Bovine leukemia virus (BLV) causes enzootic bovine leucosis. Host genetic heterozygosity at the major histocompatibility complex can enhance the ability to combat infectious diseases. However, heterozygote advantage is loci specific and depends on disease type. Bovine leukocyte antigen (BoLA)-DRB3 polymorphisms are related with BLV-infection outcome; however, whether BoLA-DRB3 heterozygotes have an advantage against BLV-induced lymphoma and proviral load (PVL) remains unclear. By analyzing 1567 BLV-infected individuals, we found that BoLA-DRB3 heterozygous status was significantly associated with lymphoma resistance irrespective of cattle breeds (p < 0.0001). Similarly, decreased PVL was observed in BoLA-DRB3 heterozygotes (p = 0.0407 for Holstein cows; p = 0.0889 for Japanese Black cattle). Our report provides first evidence of BoLA-DRB3 heterozygote advantage against BLV infection outcome.

Association of Bovine Leukemia Virus-Induced Lymphoma with BoLA-DRB3 Polymorphisms at DNA, Amino Acid, and Binding Pocket Property Levels.

Bovine leukemia virus (BLV) causes enzootic bovine leucosis, a malignant B-cell lymphoma in cattle. The DNA sequence polymorphisms of bovine leukocyte antigen (BoLA)-DRB3 have exhibited a correlation with BLV-induced lymphoma in Holstein cows. However, the association may vary between different cattle breeds. Furthermore, little is known about the relationship between BLV-induced lymphoma and DRB3 at the amino acid and structural diversity levels. Here, we comprehensively analyzed the correlation between BLV-induced lymphoma and DRB3 at DNA, amino acid, and binding pocket property levels, using 106 BLV-infected asymptomatic and 227 BLV-induced lymphoma Japanese black cattle samples. DRB3*011:01 was identified as a resistance allele, whereas DRB3*005:02 and DRB3*016:01 were susceptibility alleles. Amino acid association studies showed that positions 9, 11, 13, 26, 30, 47, 57, 70, 71, 74, 78, and 86 were associated with lymphoma susceptibility. Structure and electrostatic charge modeling further indicated that binding pocket 9 of resistance DRB3 was positively charged. In contrast, alleles susceptible to lymphoma were neutrally charged. Altogether, this is the first association study of BoLA-DRB3 polymorphisms with BLV-induced lymphoma in Japanese black cattle. In addition, our results further contribute to understanding the mechanisms regarding how BoLA-DRB3 polymorphisms mediate susceptibility to BLV-induced lymphoma.

Risk Assessment of Bovine Major Histocompatibility Complex Class II DRB3 Alleles for Perinatal Transmission of Bovine Leukemia Virus.

Perinatal transmission plays a critical role in the spread of bovine leukemia virus (BLV) infection in cattle herds. In the Holstein breed, we previously identified BLV resistant and susceptible bovine leukocyte antigen (BoLA)-DRB3 alleles, including BoLA-DRB3*009:02 and *014:01:01 with a low BLV proviral load (PVL), and *015:01 and *012:01 with a high PVL. Here, we evaluated the perinatal BLV transmission risk in dams with different BoLA-DRB3 alleles. BoLA-DRB3 alleles of 120 dam-calf pairs from five dairy farms in Japan were identified; their PVL was quantified using the BLV-Coordination of Common Motifs (CoCoMo)-qPCR-2 assay. Ninety-six dams were BLV-positive, and 29 gave birth to BLV-infected calves. Perinatal transmission frequency was 19% in dams with resistant alleles suppressed to a low PVL level, and 38% and 25% in dams with susceptible and neutral alleles that maintained high PVL levels, respectively. Notably, all calves with resistant alleles were BLV free, whereas 30% of calves with susceptible genes were infected. Thus, vertical transmission risk was extremely lower for dams and calves with resistant alleles compared to those with susceptible alleles. Our results can inform the development of effective BLV eradication programs under field conditions by providing necessary data to allow for optimal selection of dams for breeding.

Vigna radiata (L.) R. Wilczek Extract Inhibits Influenza A Virus by Targeting Viral Attachment, Penetration, Assembly, and Release.

Vigna radiata (L.) R. Wilczek (mung bean) is a Chinese functional food with antioxidant, antimicrobial and anti-inflammatory activities. However, little is known about its antiviral activity. We aimed to investigate the antiviral activity and mechanisms of action of Vigna radiata extract (VRE) against influenza virus. HPLC was conducted to analyze the components of the VRE. The anti-influenza viral activity of VRE in Mardin-Darby canine kidney (MDCK) cells was evaluated by virus titration assays, hemagglutination assays, quantitative RT-PCR assays, cellular α-glucosidase activity assays and neuraminidase activity assays. Chromatographic profiling analysis identified two major flavonoids, vitexin and isovitexin, in the ethanol extract of Vigna radiata. Through in vitro studies, we showed that VRE, at concentrations up to 2,000 µg/ml, exhibited no cytotoxicity in MDCK cells. VRE protected cells from influenza virus-induced cytopathic effects and significantly inhibited viral replication in a concentration-dependent manner. A detailed time-of-addition assay revealed that VRE may act on both the early and late stages of the viral life cycle. We demonstrated that 1) VRE inhibits virus entry by directly blocking the HA protein of influenza virus; 2) VRE inhibits virus entry by directly binding to cellular receptors; 3) VRE inhibits virus penetration; 4) VRE inhibits virus assembly by blocking cellular α-glucosidase activity, thus reducing HA protein trafficking to the cell surface; and 5) VRE inhibits virus release by inhibiting viral neuraminidase activity. In summary, Vigna radiata extract potently interferes with two different subtypes of influenza viruses at multiple steps during the infectious cycle, demonstrating its broad-spectrum potential as an anti-influenza preventive and therapeutic agent. Continued development of Vigna radiata-derived products into antiviral therapeutics is warranted.

BoLA-DRB3 Polymorphism is Associated with Differential Susceptibility to Bovine Leukemia Virus-Induced Lymphoma and Proviral Load.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. However, less than 5% of BLV-infected cattle will develop lymphoma, suggesting that, in addition to viral infection, host genetic polymorphisms might play a role in disease susceptibility. Bovine leukocyte antigen (BoLA)-DRB3 is a highly polymorphic gene associated with BLV proviral load (PVL) susceptibility. Due to the fact that PVL is positively associated with disease progression, it is believed that controlling PVL can prevent lymphoma development. Thus, many studies have focused on the relationship between PVL and BoLA-DRB3. Despite this, there is little information regarding the relationship between lymphoma and BoLA-DRB3. Furthermore, whether or not PVL-associated BoLA-DRB3 is linked to lymphoma-associated BoLA-DRB3 has not been clarified. Here, we investigated whether or not lymphoma-associated BoLA-DRB3 is correlated with PVL-associated BoLA-DRB3. We demonstrate that two BoLA-DRB3 alleles were specifically associated with lymphoma resistance (*010:01 and *011:01), but no lymphoma-specific susceptibility alleles were found; furthermore, two other alleles, *002:01 and *012:01, were associated with PVL resistance and susceptibility, respectively. In contrast, lymphoma and PVL shared two resistance-associated (DRB3*014:01:01 and *009:02) BoLA-DRB3 alleles. Interestingly, we found that PVL associated alleles, but not lymphoma associated alleles, are related with the anti-BLV gp51 antibody production level in cows. Overall, our study is the first to demonstrate that the BoLA-DRB3 polymorphism confers differential susceptibility to BLV-induced lymphoma and PVL.

Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins.

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G2/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10.

Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus.

Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.

Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A.

The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.IMPORTANCE The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.