Multiple Viral Proteins Research Articles

Phenotype and function of HBV-specific T cells is determined by the targeted epitope in addition to the stage of infection

ObjectiveChronic HBV infection affects more than 250 million people worldwide and remains a global healthcare problem in part because we lack curative treatment. Sustained viral control requires HBV-specific T cells,...

Camouflage and interception: how pathogens evade detection by intracellular nucleic acid sensors.

SummaryIntracellular DNA and RNA sensors play a vital part in the innate immune response to viruses and other intracellular pathogens, causing the secretion of type I interferons, cytokines and chemokines from infected cells. Pathogen RNA can be detected by retinoic‐acid inducible gene I‐like receptors in the cytosol, whereas cytosolic DNA is recognized by DNA sensors such as cyclic GMP‐AMP synthase (cGAS). The resulting local immune response, which is initiated within hours of infection, is able to eliminate many pathogens before they are able to establish an infection in the host. For this reason, all viruses, and some intracellular bacteria and protozoa, need to evade detection by nucleic acid sensors. Immune evasion strategies include the sequestration and modification of nucleic acids, and the inhibition or degradation of host factors involved in innate immune signalling. Large DNA viruses, such as herpesviruses, often use multiple viral proteins to inhibit signalling cascades at several different points; for instance herpes simplex virus 1 targets both DNA sensors cGAS and interferon‐γ‐inducible protein 16, as well as the adaptor protein STING (stimulator of interferon genes) and other signalling factors in the pathway. Viruses with a small genome encode only a few immunomodulatory proteins, but these are often multifunctional, such as the NS1 protein from influenza A virus, which inhibits RNA sensing in multiple ways. Intracellular bacteria and protozoa can also be detected by nucleic acid sensors. However, as the type I interferon response is not always beneficial for the host under these circumstances, some bacteria subvert, rather than evade, these signalling cascades for their own gain.

Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization.

Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans-Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization.IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.

Review of Current Cell-Penetrating Antibody Developments for HIV-1 Therapy.

The discovery of highly active antiretroviral therapy (HAART) in 1996 has significantly reduced the global mortality and morbidity caused by the acquired immunodeficiency syndrome (AIDS). However, the therapeutic strategy of HAART that targets multiple viral proteins may render off-target toxicity and more importantly results in drug-resistant escape mutants. These have been the main challenges for HAART and refinement of this therapeutic strategy is urgently needed. Antibody-mediated treatments are emerging therapeutic modalities for various diseases. Most therapeutic antibodies have been approved by Food and Drug Administration (FDA) mainly for targeting cancers. Previous studies have also demonstrated the promising effect of therapeutic antibodies against HIV-1, but there are several limitations in this therapy, particularly when the viral targets are intracellular proteins. The conventional antibodies do not cross the cell membrane, hence, the pathogenic intracellular proteins cannot be targeted with this classical therapeutic approach. Over the years, the advancement of antibody engineering has permitted the therapeutic antibodies to comprehensively target both extra- and intra-cellular proteins in various infections and diseases. This review aims to update on the current progress in the development of antibody-based treatment against intracellular targets in HIV-1 infection. We also attempt to highlight the challenges and limitations in the development of antibody-based therapeutic modalities against HIV-1.

Suppression of Adenovirus Replication by Cardiotonic Steroids.

Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

Zika virus inhibits type-I interferon production and downstream signaling.

Zika virus is an emerging mosquito-borne pathogen that is associated with Guillain-Barré syndrome in adults and microcephaly and other neurological defects in newborns. Despite being declared an international emergency by the World Health Organization, comparatively little is known about its biology. Here, we investigate the strategies employed by the virus to suppress the host antiviral response. We observe that once established, Zika virus infection is impervious to interferon treatment suggesting that the virus deploys effective countermeasures to host cell defences. This is confirmed by experiments showing that Zika virus infection impairs the induction of type-I interferon as well as downstream interferon-stimulated genes. Multiple viral proteins affect these processes. Virus-mediated degradation of STAT2 acts to reduce type-I and type-III interferon-mediated signaling. Further, the NS5 of Zika virus binds to STAT2, and its expression is correlated with STAT2 degradation by the proteasome. Together, our findings provide key insights into how Zika virus blocks cellular defense systems. This in turn is important for understanding pathogenesis and may aid in designing antiviral therapies.

Ubiquitination Upregulates Influenza Virus Polymerase Function.

The influenza A virus polymerase plays an essential role in the virus life cycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher-order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including posttranslational modifications; however, the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expression of ubiquitin upregulates polymerase function in a dose-dependent fashion, causing increased accumulation of viral RNA (vRNA), cRNA, and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of nucleoprotein (NP) or ribonucleoprotein (RNP) assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation. IMPORTANCE The cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition proviral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulation of the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.

Characterization of Vesicular Stomatitis Virus Pseudotypes Bearing Essential Entry Glycoproteins gB, gD, gH, and gL of Herpes Simplex Virus 1.

Herpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells-a prerequisite for a successful infection-is a complex process that involves multiple viral and host proteins and occurs by different routes. Detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development, yet the presence of more than a dozen proteins on the viral envelope complicates the dissection of the HSV entry mechanisms. In this study, we generated heterologous virions displaying the four essential entry proteins of HSV-1 and showed that they are capable of cell entry and, like HSV-1, require all four entry glycoproteins along with a gD receptor. This HSV pseudotyping system pioneered in this work opens doors for future systematic exploration of the herpesvirus entry mechanisms.

Integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying 18 kDa rotavirus antigen.

A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc.

Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

ABSTRACTSerial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro. Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. IMPORTANCE The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes.

Antagonising roles between PACT and influenza A virus polymerase subunits in viral replication and innate immune response

Abstract Interplay between host innate immune responses and viral antagonism determines the effectiveness of viral replication and pathogenicity. Influenza A virus (IAV) confronts host antiviral responses including type I interferon production using multiple viral proteins such as NS1. We have previously shown that double stranded RNA-binding protein PACT is an essential coactivator of RIG-I in innate antiviral response. In this study, we have further shown that PACT has dual antiviral activity in both interferon-dependent and interferon-independent manners. We found that antiviral activity of PACT is mediated through not only the activation of RIG-I and interferon production but also the direct suppression of IAV polymerase activity. We found that PACT associates with IAV polymerase subunits, namely PB2, PB1 and PA, and inhibits viral replication. On the other hand, the three polymerase subunits also counteract PACT/RIG-I-mediated production of type-I interferon. Taken together, our findings reveal a new mechanism of virus-host interaction demonstrating the mutual antagonism between PACT and IAV polymerases during IAV infection and replication. Supported by RGC (HKU1/CRF/11G, N-HKU712/12, T11-707/15-R and C7011-15R), HMRF (12111312, 14130862, HKM-15-M01 and 15140662) and S. K. Yee MRF (2011).

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.

The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection.

Large-scale production and antiviral efficacy of multi-target double-stranded RNA for the prevention of white spot syndrome virus (WSSV) in shrimp.

BackgroundRNA interference (RNAi) is a specific and effective approach for inhibiting viral replication by introducing double-stranded (ds)RNA targeting the viral gene. In this study, we employed a combinatorial approach to interfere multiple gene functions of white spot syndrome virus (WSSV), the most lethal shrimp virus, using a single-batch of dsRNA, so-called “multi-WSSV dsRNA.” A co-cultivation of RNase-deficient E. coli was developed to produce dsRNA targeting a major structural protein (VP28) and a hub protein (WSSV051) with high number of interacting protein partners.ResultsFor a co-cultivation of transformed E. coli, use of Terrific broth (TB) medium was shown to improve the growth of the E. coli and multi-WSSV dsRNA yields as compared to the use of Luria Bertani (LB) broth. Co-culture expression was conducted under glycerol feeding fed-batch fermentation. Estimated yield of multi-WSSV dsRNA (μg/mL culture) from the fed-batch process was 30 times higher than that obtained under a lab-scale culture with LB broth. Oral delivery of the resulting multi-WSSV dsRNA reduced % cumulative mortality and delayed average time to death compared to the non-treated group after WSSV challenge.ConclusionThe present study suggests a co-cultivation technique for production of antiviral dsRNA with multiple viral targets. The optimal multi-WSSV dsRNA production was achieved by the use of glycerol feeding fed-batch cultivation with controlled pH and dissolved oxygen. The cultivation technique developed herein should be feasible for industrial-scale RNAi applications in shrimp aquaculture. Interference of multiple viral protein functions by a single-batch dsRNA should also be an ideal approach for RNAi-mediated fighting against viruses, especially the large and complicated WSSV.

Deficiencies in Cellular Processes Modulated by the Retinoblastoma Protein Do Not Account for Reduced Human Cytomegalovirus Replication in Its Absence.

Despite encoding multiple viral proteins that modulate the retinoblastoma (Rb) protein in a manner classically defined as inactivation, human cytomegalovirus (HCMV) requires the presence of the Rb protein to replicate efficiently. In uninfected cells, Rb controls numerous pathways that the virus also commandeers during infection. These include cell cycle progression, senescence, mitochondrial biogenesis, apoptosis, and glutaminolysis. We investigated whether a potential inability of HCMV to regulate these Rb-controlled pathways in the absence of the Rb protein was the reason for reduced viral productive replication in Rb knockdown cells. We found that HCMV was equally able to modulate these pathways in the parental Rb-expressing and Rb-depleted cells. Our results suggest that Rb may be required to enhance a specific viral process during HCMV productive replication. The retinoblastoma (Rb) tumor suppressor is well established as a repressor of E2F-dependent transcription. Rb hyperphosphorylation, degradation, and binding by viral oncoproteins are also codified. Recent reports indicate Rb can be monophosphorylated, repress the transcription of antiviral genes in association with adenovirus E1A, modulate cellular responses to polycomb-mediated epigenetic methylations in human papillomavirus type 16 E7 expressing cells, and increase the efficiency of human cytomegalovirus (HCMV) productive replication. Since Rb function also now extends to regulation of mitochondrial function (apoptosis, metabolism), it is clear that our current understanding of this protein is insufficient to explain its roles in virus-infected cells and tumors. Work here reinforces this concept, showing the known roles of Rb are insufficient to explain its positive impact on HCMV replication. Therefore, HCMV, along with other viral systems, provide valuable tools to probe functions of Rb that might be modulated with therapeutics for cancers with viral or nonviral etiologies.

KL Vaccines against stis: where have we got to?

STIs are an increasing global challenge for public health. Infections with HSV and with drug resistant gonococci are endemic, and chlamydial infection contributes extensively to reproductive problems, while immunosuppression from HIV infection has resulted in epidemic spread of XDR Tuberculosis. Antivirals have converted HIV to a chronic disease, and have helped to control HSV. However, the underlying infections are no less frequent. Immunisation is the single most effective public health measure after safe food and water, and has resulted in control of many previously epidemic viral infections, and eradication of smallpox. The universal HPV prophylactic immunisation program in Australia, has over eight years dramatically reduced the incidence of genital warts, and of cervical pre-cancer, amongst immunised and unimmunized young Australians. However, 60 years of effort have failed to produce an effective prophylactic or therapeutic vaccine against herpes viruses, despite genetic stability and multiple immunogenic viral protein antigens. Similarly, 20 years of efforts have produced vaccines with only limited impact on prevention of HIV infection. Recent successes with vaccines effective against systemic bacterial infections offer some prospects of success for bacterial STIs, while novel vaccine technologies offer similar promise for viral STIs, though the value proposition for industry will need to be developed. The health community will need to accept vaccine programs that are not so much for individual protection as strategies to reduce the community burden of disease, and to develop effective education strategies to encourage uptake of new vaccines as they are developed.

Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

Infection of cells with DNA viruses triggers innate immune responses mediated by DNA sensors. cGMP-AMP synthase (cGAS) is a key DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which binds to and activates stimulator of interferon genes (STING), leading to IFN production and an antiviral response. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA virus that is linked to several human malignancies. We report that KSHV infection activates the cGAS-STING pathway, and that cGAS and STING also play an important role in regulating KSHV reactivation from latency. We screened KSHV proteins for their ability to inhibit this pathway and identified six viral proteins that block IFN-β activation through this pathway. This study is the first report identifying multiple viral proteins encoded by a human DNA virus that inhibit the cGAS-STING DNA sensing pathway. One such protein, viral interferon regulatory factor 1 (vIRF1), targets STING by preventing it from interacting with TANK binding kinase 1 (TBK1), thereby inhibiting STING's phosphorylation and concomitant activation, resulting in an inhibition of the DNA sensing pathway. Our data provide a unique mechanism for the negative regulation of STING-mediated DNA sensing. Moreover, the depletion of vIRF1 in the context of KSHV infection prevented efficient viral reactivation and replication, and increased the host IFN response to KSHV. The vIRF1-expressing cells also inhibited IFN-β production following infection with DNA pathogens. Collectively, our results demonstrate that gammaherpesviruses encode inhibitors that block cGAS-STING-mediated antiviral immunity, and that modulation of this pathway is important for viral transmission and the lifelong persistence of herpesviruses in the human population.

New capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines

Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID.

The human papillomavirus type 16 L1 protein directly interacts with E2 and enhances E2-dependent replication and transcription activation.

The human papillomavirus (HPV) E2 protein is a multifunctional protein essential for the control of virus gene expression, genome replication and persistence. E2 is expressed throughout the differentiation-dependent virus life cycle and is functionally regulated by association with multiple viral and cellular proteins. Here, we show for the first time to our knowledge that HPV16 E2 directly associates with the major capsid protein L1, independently of other viral or cellular proteins. We have mapped the L1 binding region within E2 and show that the α-2 helices within the E2 DNA-binding domain mediate L1 interaction. Using cell-based assays, we show that co-expression of L1 and E2 results in enhanced transcription and virus origin-dependent DNA replication. Upon co-expression in keratinocytes, L1 reduces nucleolar association of E2 protein, and when co-expressed with E1 and E2, L1 is partially recruited to viral replication factories. Furthermore, co-distribution of E2 and L1 was detected in the nuclei of upper suprabasal cells in stratified epithelia of HPV16 genome-containing primary human keratinocytes. Taken together, our findings suggest that the interaction between E2 and L1 is important for the regulation of E2 function during the late events of the HPV life cycle.

ORF45-Mediated Prolonged c-Fos Accumulation Accelerates Viral Transcription during the Late Stage of Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple viral proteins that activate extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) cascades. One of these viral proteins, ORF45, mediates sustained ERK-p90 ribosomal S6 kinase (RSK) activation during KSHV lytic replication and facilitates viral translation through the phosphorylation of a eukaryotic translation initiation factor, eIF4B. The importance of ERK-RSK activation for KSHV viral transcription has been shown; however, which transcription factor senses the sustained MAPK signaling and leads to viral transcription remains poorly understood. Here we show that the presence of ORF45 leads to the prolonged accumulation of c-Fos during the late stage of KSHV lytic replication through ERK-RSK-dependent phosphorylation and stabilization and that the depletion of c-Fos disrupts viral lytic transcription. Genome-wide screening revealed that c-Fos directly binds to multiple viral gene promoters and enhances viral transcription. Mutation of the ERK-RSK phosphorylation sites of c-Fos restrains KSHV lytic gene expression and virion production. These results indicate that the prolonged accumulation of c-Fos promotes the progression of viral transcription from early to late stages and accelerates viral lytic replication upon sustained ORF45-ERK-RSK activation during the KSHV lytic life cycle. During KSHV lytic replication, transient activation and sustained activation of ERK-RSK induce viral immediate early (IE) transcription and late transcription, respectively. Studies have revealed that ERK-RSK activates several transcription factors involved in IE gene expression, including Ets, AP-1, CREB, and C/EBP, which lead to the transient ERK-RSK activation-dependent IE transcription. Whereas c-Fos acts as a sensor of sustained ERK-RSK activation, ORF45-ERK-RSK signaling mediates c-Fos phosphorylation and accumulation during late KSHV lytic replication, consequently promoting viral transcription through the direct binding of c-Fos to multiple KSHV promoters. This finding indicates that c-Fos mediates distinct viral transcriptional progression following sustained ERK-RSK signaling during the KSHV lytic life cycle.