Infected Cell Protein Research Articles

Stress Can Induce Bovine Alpha-Herpesvirus 1 (BoHV-1) Reactivation from Latency

Bovine alpha-herpesvirus 1 (BoHV-1) is a significant problem for the cattle industry, in part because the virus establishes latency, and stressful stimuli increase the incidence of reactivation from latency. Sensory neurons in trigeminal ganglia and unknown cells in pharyngeal tonsils are importantsites for latency. Reactivation from latency can lead to reproductive problems in pregnant cows, virus transmission to young calves, suppression of immune responses, and bacterial pneumonia. BoHV-1 is also a significant cofactor in bovine respiratory disease (BRD). Stress, as mimicked by the synthetic corticosteroid dexamethasone, reproducibly initiates reactivation from latency. Stress-mediated activation of the glucocorticoid receptor (GR) stimulates viral replication and transactivation of viral promoters that drive the expression of infected cell protein 0 (bICP0) and bICP4. Notably, GR and Krüppel-like factor 15 (KLF15) form a feed-forward transcription loop that cooperatively transactivates immediate early transcription unit 1 (IEtu1 promoter). Two pioneer transcription factors, GR and KLF4, cooperatively transactivate the bICP0 early promoter. Pioneer transcription factors bind silent viral heterochromatin, remodel chromatin, and activate gene expression. Thus, wepredict that these novel transcription factors mediate early stages of BoHV-1 reactivation from latency.

Molecular characterization and phylogenetic analysis of infectious laryngotracheitis virus isolates from commercial chicken flocks in Turkey.

Infectious laryngotracheitis virus (ILTV) causes an acute and highly contagious respiratory disease in poultry. Live-attenuated vaccines are generally used to control and prevent infectious laryngotracheitis (ILT). However, these vaccines can revert to a virulent form due to multiple passages and thereby become an ILT source. Hence, monitoring of ILTV in the field through molecular characterization is critically important for controlling infection and differentiating circulating isolates. In this study, we genotypically characterized and phylogenetically analyzed eight ILTV isolates from chicken flocks located in four different cities of Turkey between 2019 and 2022. For all isolates, we analyzed two regions of the infected cell protein 4 gene (ICP4-1 and ICP4-2) and the thymidine kinase (TK) gene. The isolates were 100%, 100%, and 99.8-100% identical to each other in the ICP4-1 and ICP4-2 gene fragments and the TK gene, respectively. None of the ICP4 sequences had a deletion at nt 272-283, confirming that they were field isolates. None of the isolates were predicted to have a T252M mutation in the thymidine kinase, suggesting that they have low virulence. The isolates were 100%, 99.36%, and 99.91% identical to Turkish ILTV isolates in their ICP4-1, ICP4-2, and TK gene region, respectively. Phylogenetic analysis based on the ICP4-1 and TK genes confirmed that the ILTV isolates are closely related to Turkish ILTV isolates. This suggests that these ILTVs were endemic isolates, which in turn suggests that the ILTV isolates circulating in Turkey were evolutionarily close, originated from the field, and had low virulence.

Unraveling the assembly mechanism of SADS-CoV virus nucleocapsid protein: insights from RNA binding, dimerization, and epitope diversity profiling.

SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs.

A Broad Influenza Vaccine Based on a Heat-Activated, Tissue-Restricted Replication-Competent Herpesvirus.

Vaccination with transiently activated replication-competent controlled herpesviruses (RCCVs) expressing influenza A virus hemagglutinins broadly protects mice against lethal influenza virus challenges. The non-replicating RCCVs can be activated to transiently replicate with high efficiency. Activation involves a brief heat treatment to the epidermal administration site in the presence of a drug. The drug co-control is intended as a block to inadvertent reactivation in the nervous system and, secondarily, viremia under adverse conditions. While the broad protective effects observed raise an expectation that RCCVs may be developed as universal flu vaccines, the need for administering a co-activating drug may dampen enthusiasm for such a development. To replace the drug co-control, we isolated keratin gene promoters that were active in skin cells but inactive in nerve cells and other cells in vitro. In a mouse model of lethal central nervous system (CNS) infection, the administration of a recombinant that had the promoter of the infected cell protein 8 (ICP8) gene of a wild-type herpes simplex virus 1 (HSV-1) strain replaced by a keratin promoter did not result in any clinical signs, even at doses of 500 times wild-type virus LD50. Replication of the recombinant was undetectable in brain homogenates. Second-generation RCCVs expressing a subtype H1 hemagglutinin (HA) were generated in which the infected cell protein 4 (ICP4) genes were controlled by a heat switch and the ICP8 gene by the keratin promoter. In mice, these RCCVs replicated efficiently and in a heat-controlled fashion in the epidermal administration site. Immunization with the activated RCCVs induced robust neutralizing antibody responses against influenza viruses and protected against heterologous and cross-group influenza virus challenges.

A viral E3 ubiquitin ligase produced by herpes simplex virus 1 inhibits the NLRP1 inflammasome.

Guard proteins initiate defense mechanisms upon sensing pathogen-encoded virulence factors. Successful viral pathogens likely inhibit guard protein activity, but these interactions have been largely undefined. Here, we demonstrate that the human pathogen herpes simplex virus 1 (HSV-1) stimulates and inhibits an antiviral pathway initiated by NLRP1, a guard protein that induces inflammasome formation and pyroptotic cell death when activated. Notably, HSV-1 infection of human keratinocytes promotes posttranslational modifications to NLRP1, consistent with MAPK-dependent NLRP1 activation, but does not result in downstream inflammasome formation. We identify infected cell protein 0 (ICP0) as the critical HSV-1 protein that is necessary and sufficient for inhibition of the NLRP1 pathway. Mechanistically, ICP0's cytoplasmic localization and function as an E3 ubiquitin ligase prevents proteasomal degradation of the auto-inhibitory NT-NLRP1 fragment, thereby preventing inflammasome formation. Further, we demonstrate that inhibiting this inflammasome is important for promoting HSV-1 replication. Thus, we have established a mechanism by which HSV-1 overcomes a guard-mediated antiviral defense strategy in humans.

Involvement of SARS-CoV-2 accessory proteins in immunopathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the largest single-stranded RNA virus known to date. Its genome contains multiple accessory protein genes that act against host immune responses but are not required for progeny virus production. The functions of the accessory proteins in the viral life cycle have been examined, but their involvement in viral pathogenicity remains unclear. Here, we investigated the roles of the accessory proteins in viral immunopathogenicity. To this end,recombinant SARS-CoV-2 possessing nonsense mutations in the seven accessory protein open reading frames (ORFs) (ORF3a, ORF3b, ORF6, ORF7a, ORF8, ORF9b, and ORF10)was de novo generated using an early pandemic SARS-CoV-2 strain as a backbone. We confirmed that the resultant virus (termed ORF3-10 KO) did not express accessory proteins in infected cells and retained the desired mutations in the viral genome. In cell culture, the ORF3-10 KO virus exhibited similar virus growth kinetics as the parental virus. In hamsters, ORF3-10 KO virus infection resulted in mild weight loss and reduced viral replication in the oral cavity and lung tissue. ORF3-10 KO virus infection led to mild inflammation, indicating that an inability to evade innate immune sensing because of a lack of accessory proteins impairs virus growth in vivo and results in quick elimination from the body. Overall, we showed that SARS-CoV-2 accessory proteins are involved in immunopathogenicity.

Aberrant RNA polymerase initiation and processivity on the genome of a herpes simplex virus 1 mutant lacking ICP27.

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

A cell cycle regulator, E2F2, and glucocorticoid receptor cooperatively transactivate the bovine alphaherpesvirus 1 immediate early transcription unit 1 promoter.

Bovine alpha-herpesvirus 1 (BoHV-1) acute infection in cattle leads to establishment of latency in sensory neurons in the trigeminal ganglia (TG). A synthetic corticosteroid dexamethasone consistently initiates BoHV-1 reactivation in latently infected calves. The BoHV-1 immediate early transcription unit 1 (IEtu1) promoter regulates expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators. Hence, the IEtu1 promoter must be activated for the reactivation to occur. The number of TG neurons expressing E2F2, a transcription factor and cell cycle regulator, increased during early stages of reactivation from latency. The glucocorticoid receptor (GR) and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivated a 436-bp cis-regulatory module (CRM) in the IEtu1 promoter that contains two GR responsive elements. Chromatin immunoprecipitation studies revealed that E2F2 occupies IEtu1 promoter sequences in cultured cells. GR and E2F2 mediate cooperative transactivation of IEtu1 promoter activity, which is predicted to stimulate viral replication following stressful stimuli.

Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1–155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1).

Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1β (IL-1β) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.

Expression of the SARS-CoV-2 receptor-binding domain by live attenuated influenza vaccine virus as a strategy for designing a bivalent vaccine against COVID-19 and influenza

Influenza and SARS-CoV-2 are two major respiratory pathogens that cocirculate in humans and cause serious illness with the potential to exacerbate disease in the event of co-infection. To develop a bivalent vaccine, capable of protecting against both infections, we inserted the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein into hemagglutinin (HA) molecule or into the open reading frame of the truncated nonstructural protein 1 (NS1) of live attenuated influenza vaccine (LAIV) virus and assessed phenotypic characteristics of the rescued LAIV-RBD viruses, as well as their immunogenicity in mouse and Syrian hamster animal models. A panel of 9 recombinant LAIV-RBD viruses was rescued using the A/Leningrad/17 backbone. Notably, only two variants with RBD insertions into the HA molecule could express sufficient quantities of RBD protein in infected MDCK cells. Intranasal immunization of mice induced high levels of anti-influenza antibody responses in all chimeric LAIV-RBD viruses, which was comparable to the LAIV virus vector. The RBD-specific antibody responses were most pronounced in the variant expressing RBD194 fragment as a chimeric HA protein. This candidate was further tested in Syrian hamsters and was shown to be immunogenic and capable of protecting animals against both infections.

Circulation and Molecular Characterization of Infectious Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Turkey, 2018-2022.

Infectious laryngotracheitis (ILT) is a very serious worldwide respiratory disease of poultry, with many countries reporting ILT infections over the last decade. However, few reports are available regarding ILT disease prevalence in poultry in Turkey. Accordingly, the present study investigated ILT infection in Turkish broiler flocks between 2018 and 2022. Circulating ILT strains were characterized by sequence and phylogenetic analysis of two fragments of the infected-cell protein 4 gene. ILT virus (ILTV) was confirmed by quantitative PCR in 8 of the 21 flocks examined. As in other diseases, co-infections with other respiratory pathogens in confirmed ILT cases may worsen the symptoms and prolong the disease course. The present study confirmed co-infections with infectious bronchitis virus (13/21 tested flocks and 5/8 ILTV-positive flocks), indicating the importance of these pathogens in the occurrence of ILT infections.

Effect of Melatonin on Herpesvirus Type 1 Replication.

Acute HSV-1 infection is associated with mild symptoms, such as fever and lesions of the mouth, face and skin. This phase is followed by a latency period before reactivation, which is associated with symptoms ranging from ulcers to encephalitis. Despite available anti-HSV-1 drugs, the development of new antiviral agents is sought due to the presence of resistant viruses. Melatonin, a molecule secreted by the pineal gland, has been shown to be an antioxidant, inducer of antioxidant enzymes, and regulator of various biological processes. Clinical trials have explored its therapeutic utility in conditions including infections. This study focuses on melatonin's role in HSV-1 replication and the underlying mechanisms. Melatonin was found to decrease the synthesis of HSV-1 proteins in infected Vero cells measured by immunofluorescence, indicating an inhibition of HSV-1 replication. Additionally, it regulates the activities of antioxidant enzymes and affects proteasome activity. Melatonin activates the unfolded protein response (UPR) and autophagy and suppresses apoptosis in HSV-1-infected cells. In summary, melatonin demonstrates an inhibitory role in HSV-1 replication by modulating various cellular responses, suggesting its potential utility in the treatment of viral infections.

Abstract 6656: Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus

Abstract Major challenges for CAR-T cell therapy in solid tumors include various limitations due to the issue of appropriate antigen selection, tumor heterogeneity, and the immune-suppressive tumor microenvironment (TME). To overcome these hurdles, we’ve developed a novel therapy, CLM-202 which is a combination therapy of oncolytic Newcastle disease virus (NDV) and NDV F protein-targeted CAR-T cell therapy (NDV F CAR-T). After NDV infects tumor cells, NDV envelope protein F is expressed on the tumor cell surface. NDV F CAR-T recognizes NDV F protein and kills the tumor cells. This approach can be applied to patients with various indications, regardless of the presence of the target protein. Using NDV to deliver antigens minimizes the risk of tumor recurrence due to tumor heterogeneity. Furthermore, it mitigates off-target toxicity by targeting oncolytic virus-derived proteins, not human proteins. Oncolytic NDV also triggers a potent immune response within the tumor microenvironment, thereby altering the immunosuppressive conditions of the tumor and improving CAR-T efficacy. Therefore, CLM-202 can be a multipurpose solution for solid tumors. We have developed a second-generation CAR-T cell therapy, NDV F CAR-T, which can be used in combination with an oncolytic virus known as NDV. NDV F CAR-T is designed to specifically target the F protein expressed when cells are infected by NDV. It incorporates a single-chain variable fragment (scFv) derived from an antibody that can bind specifically to the F protein of NDV-infected cells. To assess the activity of NDV F CAR-T in tumor cells against a variety of solid tumors as indications, we conducted several experiments. We initially confirmed the expression of the F protein in NDV-infected tumor cells. We also conducted cytotoxicity tests to confirm NDV F CAR-T's ability to induce death in NDV-infected tumor cell lines. Furthermore, to assess the efficacy of NDV F CAR-T in an in vivo model, we established an orthotopic mouse model. In this model, we administered NDV via intratumoral injection, followed by the single-dose administration of NDV F CAR-T to perform the efficacy test. We have confirmed that NDV infects all solid tumor cell lines representing different indications and induces the expression of the F protein in infected tumor cells. Furthermore, we have observed that NDV infection in these diverse tumor cells results in robust tumor cell-killing activity by NDV F CAR-T and the subsequent activation of CAR-T. Moreover, in an orthotopic mouse model, we have demonstrated that the administration of NDV followed by NDV F CAR-T induces potent tumor growth inhibition. In summary, our research proposes a novel therapeutic strategy, CLM-202 that utilizes NDV F CAR-T and NDV to overcome the limitations of CAR-T cell therapy in solid tumors, including tumor heterogeneity and immune-suppressive tumor microenvironments. This approach aims to effectively treat a wide range of indications. Citation Format: Soyoung Choi, Kiwan Kim, Sangeun Lee, Jeongeun Choi, Song-Jae Lee, Yong Gu Lee, Jeonghun Kim, Hoyoung Lee, Kisoon Kim, Man-Seong Park, Seong-Won Song. Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6656.

Comparison of the oncolytic activity of a replication-competent and a replication-deficient herpes simplex virus 1.

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-β mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.

HSV-1 ICP0 dimer domain adopts a novel β-barrel fold.

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine β-strands and two α-helices. Interestingly, two adjacent β-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two β-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two β-strands of each dimer, creating a "stacking" of the β-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

An Improved Workflow for the Quantification of Orthohantavirus Infection Using Automated Imaging and Flow Cytometry.

Determination of the infectious titer is a central requirement when working with pathogenic viruses. The plaque or focus assay is a commonly used but labor- and time-consuming approach for determining the infectious titer of orthohantavirus samples. We have developed an optimized virus quantification approach that relies on the fluorescence-based detection of the orthohantavirus nucleocapsid protein (N) in infected cells with high sensitivity. We present the use of flow cytometry but highlight fluorescence microscopy in combination with automated data analysis as an attractive alternative to increase the information retrieved from an infection experiment. Additionally, we offer open-source software equipped with a user-friendly graphical interface, eliminating the necessity for advanced programming skills.

GSK-3β as a Potential Coordinator of Anabolic and Catabolic Pathways in Hepatitis C Virus Insulin Resistance

Introduction: Chronic hepatitis C infection can result in insulin resistance (IR). We have previously shown that it occurs through the interaction of pathways for glucose homeostasis, insulin signaling, and autophagy. But it is not known how soon the pathways are activated and how IR is related to the signals generated by catabolic and anabolic conditions occurring in infected cells. We have extended our studies to a cell culture system mimicking acute infection and to downstream pathways involving energy-sensor AMPK and nutrient-sensor mTOR that are active in catabolic and anabolic processes within the infected cells. Methods: Huh7 liver cells in culture were infected with hepatitis C virus (HCV). We performed proteomics analysis of key proteins in infected cells by Western blotting and IP experiments, with or without IFNα exposure as a component of conventional therapeutic strategy. Results: We present evidence that (a) IRS-1 Ser312, Beclin-1, protein conjugate Atg12-Atg5 or GS Ser641 are up-regulated early in infection presumably by activating the same pathways as utilized for persistent infection; (b) Bcl-XL, an inhibitor of both autophagy and apoptosis, is present in a core complex with IRS-1 Ser312 and Beclin-1 during progression of IR; (c) AMPK level remains about the same in infected cells where it is activated by phosphorylation at Thr172 concomitant with increased autophagy, a hallmark of catabolic conditions; (d) an mTOR level that promotes anabolism is increased rather than decreased under an expanded autophagy; (e) hypophosphorylation of translational repressor 4E-BP1 downstream of mTOR is suggestive of reduced protein synthesis; and (f) β-catenin, is up-regulated but not phosphorylated suggesting indirectly our previous contention that its kinase, GSK-3β, is mostly in an inactive state. Conclusion: We report that in the development of IR following chronic infection, anabolic and catabolic pathways are activated early, and the metabolic interaction occurs possibly in a core complex with IRS-1 Ser312, Beclin-1, and autophagy inhibitor Bcl-XL. Induction of autophagy is usually controlled by a two-edged mechanism acting in opposition under anabolic and catabolic conditions by AMPK/mTOR/4E-BP1 pathway with GSK-3β-mediated feedback loops. However, we have observed an up-regulation of mTOR along with an up-regulation of AMPK caused by HCV infection is a deviation from the normal scenario described above which might be of therapeutic interest.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

Identification of the protein coding capability of coronavirus defective viral genomes by mass spectrometry

During coronavirus infection, in addition to the well-known coronavirus genomes and subgenomic mRNAs, an abundance of defective viral genomes (DVGs) can also be synthesized. In this study, we aimed to examine whether DVGs can encode proteins in infected cells. Nanopore direct RNA sequencing and liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis were employed. With the protein databases generated by nanopore direct RNA sequencing and the cell lysates derived from the RNA–protein pull-down assay, six DVG-encoded proteins were identified by LC–MS/MS based on the featured fusion peptides caused by recombination during DVG synthesis. The results suggest that the coronavirus DVGs have the capability to encode proteins. Consequently, future studies determining the biological function of DVG-encoded proteins may contribute to the understanding of their roles in coronavirus pathogenesis and the development of antiviral strategies.