Simian varicella virus infection and reactivation in rhesus macaques trigger cytokine and Aß40/42 alterations in serum and cerebrospinal fluid.

Simian varicella virus (SVV) produces peripheral inflammatory responses during varicella (primary infection) and zoster (reactivation) in rhesus macaques (RM). However, it is unclear if peripheral measures are accurate proxies for central nervous system (CNS) responses. Thus, we analyzed cytokine and Aß42/Aß40 changes in paired serum and cerebrospinal fluid (CSF) during the course of infection. During varicella and zoster, every RM had variable changes in serum and CSF cytokine and Aß42/Aß40 levels compared to pre-inoculation levels. Overall, peripheral infection appears to affect CNS cytokine and Aß42/Aß40 levels independent of serum responses, suggesting that peripheral disease may contribute to CNS disease.

c-Jun signaling during initial HSV-1 infection modulates latency to enhance later reactivation in addition to directly promoting the progression to full reactivation.

Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates to permit transmission, which can result in clinical manifestations. Viral transactivators required for lytic infection are largely absent during latent infection, and therefore, HSV-1 relies on the co-option of neuronal host signaling pathways to initiate its gene expression. The activation of the neuronal c-Jun N-terminal kinase (JNK) cell stress pathway is central to initiating biphasic reactivation in response to multiple stimuli. However, how host factors work with JNK to stimulate the initial wave of gene expression (known as Phase I) or the progression to full Phase II reactivation remains unclear. Here, we found that c-Jun, the primary target downstream of neuronal JNK cell stress signaling, functions during reactivation but not during the JNK-mediated initiation of Phase I gene expression. Instead, c-Jun was required to transition from Phase I to full HSV-1 reactivation and was detected in viral replication compartments of reactivating neurons. Interestingly, we also identified a role for both c-Jun and enhanced neuronal stress during initial neuronal infection in promoting a more reactivation-competent form of HSV-1 latency. Therefore, c-Jun functions at multiple stages during the HSV latent infection of neurons to promote reactivation but not during the initial JNK-dependent Phase I. Importantly, by demonstrating that initial infection conditions can contribute to later reactivation abilities, this study highlights the potential for latently infected neurons to maintain a molecular scar of previous exposure to neuronal stressors.IMPORTANCEThe molecular mechanisms that regulate the reactivation of herpes simplex virus-1 (HSV-1) from latent infection are unknown. The host transcription and pioneer factor c-Jun is the main target of the JNK cell stress pathway that is known to be important in exit of HSV from latency. Surprisingly, we found that c-Jun does not act with JNK during exit from latency but instead promotes the transition to full reactivation. Moreover, c-Jun and enhanced neuronal stress during initial neuronal infection promoted a more reactivation-competent form of HSV-1 latency. c-Jun, therefore, functions at multiple stages during HSV-1 latent infection of neurons to promote reactivation. Importantly, this study contributes to a growing body of evidence that de novo HSV-1 infection conditions can modulate latent infection and impact future reactivation events, raising important questions on the clinical impact of stress during initial HSV-1 acquisition on future reactivation events and consequences.

B cell expression of E3 ubiquitin ligase Cul4b promotes chronic gammaherpesvirus infection in vivo.

IMPORTANCE: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

Rewiring of the Host Cell Metabolome and Lipidome during Lytic Gammaherpesvirus Infection Is Essential for Infectious-Virus Production.

Oncogenic virus infections are estimated to cause ~15% of all cancers. Two prevalent human oncogenic viruses are members of the gammaherpesvirus family: Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV). We use murine herpesvirus 68 (MHV-68), which shares significant homology with KSHV and EBV, as a model system to study gammaherpesvirus lytic replication. Viruses implement distinct metabolic programs to support their life cycle, such as increasing the supply of lipids, amino acids, and nucleotide materials necessary to replicate. Our data define the global changes in the host cell metabolome and lipidome during gammaherpesvirus lytic replication. Our metabolomics analysis found that MHV-68 lytic infection induces glycolysis, glutaminolysis, lipid metabolism, and nucleotide metabolism. We additionally observed an increase in glutamine consumption and glutamine dehydrogenase protein expression. While both glucose and glutamine starvation of host cells decreased viral titers, glutamine starvation led to a greater loss in virion production. Our lipidomics analysis revealed a peak in triacylglycerides early during infection and an increase in free fatty acids and diacylglyceride later in the viral life cycle. Furthermore, we observed an increase in the protein expression of multiple lipogenic enzymes during infection. Interestingly, pharmacological inhibitors of glycolysis or lipogenesis resulted in decreased infectious virus production. Taken together, these results illustrate the global alterations in host cell metabolism during lytic gammaherpesvirus infection, establish essential pathways for viral production, and recommend targeted mechanisms to block viral spread and treat viral induced tumors. IMPORTANCE Viruses are intracellular parasites which lack their own metabolism, so they must hijack host cell metabolic machinery in order to increase the production of energy, proteins, fats, and genetic material necessary to replicate. Using murine herpesvirus 68 (MHV-68) as a model system to understand how similar human gammaherpesviruses cause cancer, we profiled the metabolic changes that occur during lytic MHV-68 infection and replication. We found that MHV-68 infection of host cells increases glucose, glutamine, lipid, and nucleotide metabolic pathways. We also showed inhibition or starvation of glucose, glutamine, or lipid metabolic pathways results in an inhibition of virus production. Ultimately, targeting changes in host cell metabolism due to viral infection can be used to treat gammaherpesvirus-induced cancers and infections in humans.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

Slug, a Stress-Induced Transcription Factor, Stimulates Herpes Simplex Virus 1 Replication and Transactivates a cis-Regulatory Module within the VP16 Promoter.

Stress-mediated activation of the glucocorticoid receptor (GR) and specific stress-induced transcription factors stimulate herpes simplex virus 1 (HSV-1) productive infection, explant-induced reactivation, and immediate early (IE) promoters that drive expression of infected cell protein 0 (ICP0), ICP4, and ICP27. Several published studies concluded the virion tegument protein VP16, ICP0, and/or ICP4 drives early steps of reactivation from latency. Notably, VP16 protein expression was induced in trigeminal ganglionic neurons of Swiss Webster or C57BL/6J mice during early stages of stress-induced reactivation. If VP16 mediates reactivation, we hypothesized stress-induced cellular transcription factors would stimulate its expression. To address this hypothesis, we tested whether stress-induced transcription factors transactivate a VP16 cis-regulatory module (CRM) located upstream of the VP16 TATA box (-249 to -30). Initial studies revealed the VP16 CRM cis-activated a minimal promoter more efficiently in mouse neuroblastoma cells (Neuro-2A) than mouse fibroblasts (NIH-3T3). GR and Slug, a stress-induced transcription factor that binds enhancer boxes (E-boxes), were the only stress-induced transcription factors examined that transactivated the VP16 CRM construct. GR- and Slug-mediated transactivation was reduced to basal levels when the E-box, two 1/2 GR response elements (GREs), or NF-κB binding site was mutated. Previous studies revealed GR and Slug cooperatively transactivated the ICP4 CRM, but not ICP0 or ICP27. Silencing of Slug expression in Neuro-2A cells significantly reduced viral replication, indicating Slug-mediated transactivation of ICP4 and VP16 CRM activity correlates with enhanced viral replication and reactivation from latency. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in several types of neurons. Periodically cellular stressors trigger reactivation from latency. Viral regulatory proteins are not abundantly expressed during latency, indicating cellular transcription factors mediate early stages of reactivation. Notably, the glucocorticoid receptor (GR) and certain stress-induced transcription factors transactivate cis-regulatory modules (CRMs) essential for expression of infected cell protein 0 (ICP0) and ICP4, key viral transcriptional regulatory proteins linked to triggering reactivation from latency. Virion protein 16 (VP16) specifically transactivates IE promoter and was also reported to mediate early stages of reactivation from latency. GR and Slug, a stress-induced enhancer box (E-box) binding protein, transactivate a minimal promoter downstream of VP16 CRM, and these transcription factors occupy VP16 CRM sequences in transfected cells. Notably, Slug stimulates viral replication in mouse neuroblastoma cells suggesting Slug, by virtue of transactivating VP16 and ICP4 CRM sequences, can trigger reactivation in certain neurons.

Characterization of a Unique Novel LORF3 Protein of Duck Plague Virus and Its Potential Pathogenesis.

Duck plague virus (DPV) is a high-morbidity fowl alphaherpesvirus that causes septicemic lesions in various organs. Most DPV genes are conserved among herpesviruses, while a few are specific to fowl herpesviruses, including the LORF3 gene, for which there is currently no literature describing its biological properties and functions. This study first addressed whether the LORF3 protein is expressed by making specific polyclonal antibodies. We could demonstrate that DPV LORF3 is an early gene and encodes a protein involved in virion assembly, mainly localized in the nucleus of DPV-infected DEF cells. To investigate the role of this novel LORF3 protein in DPV pathogenesis, we generated a recombinant virus that lacks expression of the LORF3 protein. Our data revealed that the LORF3 protein is not essential for viral replication but contributes to DPV replication in vitro and in vivo and promotes duck plague disease morbidity and mortality. Interestingly, deletion of the LORF3 protein abolished thymus atrophy in DPV-vaccinated ducks. In conclusion, this study revealed the expression of avian herpesviruses-specific genes and unraveled the role of the early protein LORF3 in the pathogenesis of DPV. IMPORTANCE DPV is a highly lethal alphaherpesvirus that causes duck plague in birds of the order Anseriformes. The virus has caused huge economic losses to the poultry industry due to high morbidity and mortality and the cost of vaccination. DPV encodes 78 open reading frames (ORFs), and these genes are involved in various processes of the viral life cycle. Functional characterization of DPV genes is important for understanding the complex viral life cycle and DPV pathogenesis. Here, we identified a novel protein encoded by LORF3, and our data suggest that the LORF3 protein is involved in the occurrence and development of duck plague.

Kaposi's Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient KSHV (21KD) and stop codon-induced ORF21-deleted KSHV (21del). The results showed that both ORF21 mutations did not affect viral genome replication, lytic gene transcription, or the production of viral genome-encapsidated particles. The ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of the progeny virus. ORF21 was expressed 36 h after the induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm. Moreover, ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. The inhibition of MEK signaling by U0126 in recipient target cells suppressed the number of progeny virus-infected cells. These suggest that ORF21 transmitted as a tegument protein in the progeny virus enhances the new infection through MEK up-regulation in the recipient cell. Our findings indicate that ORF21 plays key roles in the infection of KSHV through the manipulation of the cellular function.

The Contribution of Kaposi's Sarcoma-Associated Herpesvirus ORF7 and Its Zinc-Finger Motif to Viral Genome Cleavage and Capsid Formation.

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in alpha- and betaherpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring nonspherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Interestingly, ORF17 function was also necessary for TR cleavage. Thus, our data revealed ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCE In herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficient KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.

Gammaherpesvirus-mediated repression reveals EWSR1 to be a negative regulator of B cell responses.

The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.

T Cell-Intrinsic Interleukin 17 Receptor A Signaling Supports the Establishment of Chronic Murine Gammaherpesvirus 68 Infection.

Gammaherpesviruses, such as human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), are species-specific, ubiquitous pathogens that are associated with multiple cancers, including B cell lymphomas. These viruses have a natural tropism for B cells and usurp B cell differentiation to drive a unique and robust polyclonal germinal center response to establish a long-term latent reservoir in memory B cells. The robust polyclonal germinal center response driven by gammaherpesvirus infection increases the risk for B cell transformation. Unsurprisingly, many gammaherpesvirus cancers are derived from germinal center or post-germinal center B cells. The viral and host factors that influence the gammaherpesvirus-driven germinal center response are not clearly defined. We previously showed that host interleukin 17 receptor A (IL-17RA) signaling promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we found that T cell-intrinsic IL-17RA signaling recapitulates some proviral aspects of global IL-17RA signaling during MHV68 infection. Specifically, we found that T cell-intrinsic IL-17RA signaling supports the MHV68-driven germinal center response, the establishment of latency in the spleen, and viral reactivation in the spleen and peritoneal cavity. Our study unveils an unexpected finding where the T cell-specific IL-17RA signaling supports the establishment of a latent reservoir of a B cell-tropic gammaherpesvirus. IMPORTANCE Gammaherpesviruses, such as human EBV, establish lifelong infection in >95% of adults and are associated with B cell lymphomas. Gammaherpesviruses usurp the germinal center response to establish latent infection, and the germinal center B cells are thought to be the target of viral transformation. We previously found that global expression of IL-17RA promotes the establishment of chronic MHV68 infection and the MHV68-driven germinal center response. In this study, we showed that T cell-intrinsic IL-17RA signaling is necessary to promote the MHV68-driven germinal center response by supporting CD4+ T follicular helper cell expansion. We also found that T cell-intrinsic IL-17RA signaling contributes to but is not solely responsible for the systemic proviral role of IL-17RA signaling, highlighting the multifaceted function of IL-17RA signaling during MHV68 infection.

Lytic Replication and Reactivation from B Cells Is Not Required for Establishing or Maintaining Gammaherpesvirus Latency In Vivo.

Gammaherpesviruses (GHVs) are lymphotropic tumor viruses with a biphasic infectious cycle. Lytic replication at the primary site of infection is necessary for GHVs to spread throughout the host and establish latency in distal sites. Dissemination is mediated by infected B cells that traffic hematogenously from draining lymph nodes to peripheral lymphoid organs, such as the spleen. B cells serve as the major reservoir for viral latency, and it is hypothesized that periodic reactivation from latently infected B cells contributes to maintaining long-term chronic infection. While fundamentally important to an understanding of GHV biology, aspects of B cell infection in latency establishment and maintenance are incompletely defined, especially roles for lytic replication and reactivation in this cell type. To address this knowledge gap and overcome limitations of replication-defective viruses, we generated a recombinant murine gammaherpesvirus 68 (MHV68) in which ORF50, the gene that encodes the essential immediate-early replication and transcription activator protein (RTA), was flanked by loxP sites to enable conditional ablation of lytic replication by ORF50 deletion in cells that express Cre recombinase. Following infection of mice that encode Cre in B cells with this virus, splenomegaly and viral reactivation from splenocytes were significantly reduced; however, the number of latently infected splenocytes was equivalent to WT MHV68. Despite ORF50 deletion, MHV68 latency was maintained over time in spleens of mice at levels approximating WT, reactivation-competent MHV68. Treatment of infected mice with lipopolysaccharide (LPS), which promotes B cell activation and MHV68 reactivation ex vivo, yielded equivalent increases in the number of latently infected cells for both ORF50-deleted and WT MHV68, even when mice were simultaneously treated with the antiviral drug cidofovir to prevent reactivation. Together, these data demonstrate that productive viral replication in B cells is not required for MHV68 latency establishment and support the hypothesis that B cell proliferation facilitates latency maintenance in vivo in the absence of reactivation. IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system and place infected hosts at risk for developing lymphomas and other diseases. It is hypothesized that gammaherpesviruses must initiate acute infection in these cells to establish and maintain long-term infection, but this has not been directly tested. We report here the use of a viral genetic system that allows for cell-type-specific deletion of a viral gene that is essential for replication and reactivation. We employ this system in an in vivo model to reveal that viral replication is not required to initiate or maintain infection within B cells.

The m6A reader YTHDC2 is essential for escape from KSHV SOX-induced RNA decay.

The role of N6-methyladenosine (m6A) modifications has increasingly been associated with a diverse set of roles in modulating viruses and influencing the outcomes of viral infection. Here, we report that the landscape of m6A deposition is drastically shifted during Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection for both viral and host transcripts. In line with previous reports, we also saw an overall decrease in host methylation in favor of viral messenger RNA (mRNA), along with 5' hypomethylation and 3' hypermethylation. During KSHV lytic infection, a major shift in overall mRNA abundance is driven by the viral endoribonuclease SOX, which induces the decay of greater than 70% of transcripts. Here, we reveal that interlukin-6 (IL-6) mRNA, a well-characterized, SOX-resistant transcript, is m6A modified during lytic infection. Furthermore, we show that this modification falls within the IL-6 SOX resistance element, an RNA element in the IL-6 3' untranslated region (UTR) that was previously shown to be sufficient for protection from SOX cleavage. We show that the presence of this m6A modification is essential to confer SOX resistance to the IL-6 mRNA. We next show that this modification recruits the m6A reader YTHDC2 and found that YTHDC2 is necessary for the escape of the IL-6 transcript. These results shed light on how the host cell has evolved to use RNA modifications to circumvent viral manipulation of RNA fate during KSHV infection.

H84T BanLec has broad spectrum antiviral activity against human herpesviruses in cells, skin, and mice.

H84T BanLec is a molecularly engineered lectin cloned from bananas with broad-spectrum antiviral activity against several RNA viruses. H84T BanLec dimers bind glycoproteins containing high-mannose N-glycans on the virion envelope, blocking attachment, entry, uncoating, and spread. It was unknown whether H84T BanLec is effective against human herpesviruses varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and herpes simplex virus 1 (HSV-1), which express high-mannose N-linked glycoproteins on their envelopes. We evaluated H84T BanLec against VZV-ORF57-Luc, TB40/E HCMV-fLuc-eGFP, and HSV-1 R8411 in cells, skin organ culture, and mice. The H84T BanLec EC50 was 0.025 µM for VZV (SI50 = 4000) in human foreskin fibroblasts (HFFs), 0.23 µM for HCMV (SI50 = 441) in HFFs, and 0.33 µM for HSV-1 (SI50 = 308) in Vero cells. Human skin was obtained from reduction mammoplasties and prepared for culture. Skin was infected and cultured up to 14 days. H84T BanLec prevented VZV, HCMV and HSV-1 spread in skin at 10 µM in the culture medium, and also exhibited dose-dependent antiviral effects. Additionally, H84T BanLec arrested virus spread when treatment was delayed. Histopathology of HCMV-infected skin showed no overt toxicity when H84T BanLec was present in the media. In athymic nude mice with human skin xenografts (NuSkin mice), H84T BanLec reduced VZV spread when administered subcutaneously prior to intraxenograft virus inoculation. This is the first demonstration of H84T BanLec effectiveness against DNA viruses. H84T BanLec may have additional unexplored activity against other, clinically relevant, glycosylated viruses.

Whole-genome sequencing of Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) reveals evidence for two African lineages.

Kaposi sarcoma (KS)-associated herpesvirus (KSHV/HHV-8) was first sequenced from the body cavity (BC) lymphoma cell line, BC-1, in 1996. Few other KSHV genomes have been reported. Our knowledge of sequence variation for this virus remains spotty. This study reports additional genomes from historical US patient samples and from African KS biopsies. It describes an assay that spans regions of the virus that cannot be covered by short read sequencing. These include the terminal repeats, the LANA repeats, and the origins of replication. A phylogenetic analysis, based on 107 genomes, identified three distinct clades; one containing isolates from USA/Europe/Japan collected in the 1990s and two of Sub-Saharan Africa isolates collected since 2010. This analysis indicates that the KSHV strains circulating today differ from the isolates collected at the height of the AIDS epidemic. This analysis helps experimental designs and potential vaccine studies.

Effects of between and within Herd Moves on Elephant Endotheliotropic Herpesvirus (EEHV) Recrudescence and Shedding in Captive Asian Elephants (Elephas maximus).

Haemorrhagic disease associated with elephant endotheliotropic herpesvirus (Elephantid herpesvirus, EEHV) infections is the leading cause of death for Asian elephant (Elephas maximus) calves. This study assessed the effect of captive herd management on EEHV shedding, as evidence of latent infection reactivation, focusing on: (1) the influence of social change on the odds of recrudescence; (2) the respective effects of between and within herd moves; and (3) characteristics of recrudescent viral shedding. Trunk and conjunctival swabs (n = 165) were obtained from six elephants at an EAZA-accredited zoo, collected during a period of social stability, and at times of social change. Longitudinal sampling took place at times of moving two bulls out of the collection and one new bull into an adjacent enclosure to the cow herd (between herd moves), and during a period of mixing this new bull with the cow herd to facilitate mating (within herd moves). Quantitative PCR was employed to detect EEHV 1a/b, 4a/b, and EF-1-α (housekeeping gene). Generalised estimating equations determined EEHV recrudescence odds ratios (OR) and relative viral DNA load. Sixteen EEHV 1a/b shedding events occurred, but no EEHV 4a/b was detected. All management-derived social changes promoted recrudescence (social change OR = 3.27, 95% CI = 0.412-26, p = 0.262; and between herd moves OR = 1.6, 95% CI = 0.178-14.4, p = 0.675), though within herd movements posed the most significant increase of EEHV reactivation odds (OR = 6.86, 95% CI = 0.823-57.1, p = 0.075) and demonstrated the strongest relative influence (post hoc Tukey test p = 0.0425). Shedding onset and magnitude ranged from six to 54 days and from 3.59 to 11.09 ΔCts. Differing challenges are associated with between and within herd movements, which can promote recrudescence and should be considered an exposure risk to naïve elephants.

Elephant Endotheliotropic Herpesvirus 1, 4 and 5 in China: Occurrence in Multiple Sample Types and Implications for Wild and Captive Population Surveillance.

Elephant endotheliotropic herpesviruses (EEHVs) are important causes of death in both captive and wild Asian elephants (Elephas maximus). Nothing is known about the prevalence of EEHVs in wild or domestic elephants in China. To determine if EEHVs are present in elephants in China, 126 wild elephants from three populations and 202 captive individuals from zoos (n = 155) and the Wild Elephant Valley (n = 47) were screened using semi-nested polymerase chain reaction assays with EEHV-redundant and EEHV1/4/5-specific primers. EEHV1B and EEHV4 were detected in samples from both wild (EEHV1B:8/126; EEHV4:2/126) and captive (EEHV1B:5/155; EEHV4:9/155) elephants, while EEHV1A (six cases) and EEHV5 (one case) were only present in the captive elephants from the Wild Elephant Valley. EEHV1 was detected in blood and trunk and oral swabs; EEHV4 was detected in trunk and oral swabs as well as feces; EEHV5 was found in trunk and oral swabs. No significant age or sex association with EEHV1A, EEHV1B, or EEHV5 positivity was observed. An age association with EEHV4 positivity was found, with all unweaned elephants being EEHV4 positive, but an association with the sex of the elephant was not observed. These findings represent the first documentation of EEHV presence in captive and wild elephants in China. These findings also document EEHV1B and EEHV4 shedding in feces and demonstrate the utility of fecal screening as a tool for investigating EEHV4 infection in wild populations of elephants. It is recommended that EEHV testing be included in surveillance programs for captive and wild elephants in China.

Caspase-Mediated Cleavage of the Transcription Factor Sp3: Possible Relevance to Cancer and the Lytic Cycle of Kaposi's Sarcoma-Associated Herpesvirus.

The open reading frame 50 (ORF50) protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the master regulator essential for initiating the viral lytic cycle. Previously, we have demonstrated that the ORF50 protein can cooperate with Sp3 to synergistically activate a set of viral and cellular gene promoters through highly conserved ORF50-responsive elements that harbor a Sp3-binding motif. Herein, we show that Sp3 undergoes proteolytic cleavage during the viral lytic cycle, and the cleavage of Sp3 is dependent on caspase activation. Since similar cleavage patterns of Sp3 could be detected in both KSHV-positive and KSHV-negative lymphoma cells undergoing apoptosis, the proteolytic cleavage of Sp3 could be a common event during apoptosis. Mutational analysis identifies 12 caspase cleavage sites in Sp3, which are situated at the aspartate (D) positions D17, D19, D180, D273, D275, D293, D304 (or D307), D326, D344, D530, D543, and D565. Importantly, we noticed that three stable Sp3 C-terminal fragments generated through cleavage at D530, D543, or D565 encompass an intact DNA-binding domain. Like the full-length Sp3, the C-terminal fragments of Sp3 could still retain the ability to cooperate with ORF50 protein to activate specific viral and cellular gene promoters synergistically. Collectively, our findings suggest that despite the proteolytic cleavage of Sp3 under apoptotic conditions, the resultant Sp3 fragments may retain biological activities important for the viral lytic cycle or for cellular apoptosis. IMPORTANCE The ORF50 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the key viral protein that controls the switch from latency to lytic reactivation. It is a potent transactivator that can activate target gene promoters via interacting with other cellular DNA-binding transcription factors, such as Sp3. In this report, we show that Sp3 is proteolytically cleaved during the viral lytic cycle, and up to 12 caspase cleavage sites are identified in Sp3. Despite the proteolytic cleavage of Sp3, several resulting C-terminal fragments that have intact zinc-finger DNA-binding domains still retain substantial influence in the synergy with ORF50 to activate specific gene promoters. Overall, our studies elucidate the caspase-mediated cleavage of Sp3 and uncover how ORF50 utilizes the cleavage fragments of Sp3 to transactivate specific viral and cellular gene promoters.

Progesterone Sporadically Induces Reactivation from Latency in Female Calves but Proficiently Stimulates Bovine Herpesvirus 1 Productive Infection.

Acute infection of the ocular, oral, or nasal cavity by bovine herpesvirus 1 (BoHV-1) culminates in lifelong latency in sensory neurons within trigeminal ganglia. The BoHV-1 latency reactivation cycle, including calves latently infected with commercially available modified live vaccines, can lead to reproductive complications, including abortions. Recent studies demonstrated progesterone stimulated BoHV-1 productive infection and sporadically induced reactivation from latency in male rabbits. The progesterone receptor (PR) and progesterone transactivate the immediate early transcription unit 1 (IEtu1) promoter and the infected cell protein 0 (bICP0) early promoter. These viral promoters drive expression of two viral transcriptional regulatory proteins (bICP0 and bICP4) that are crucial for productive infection. Based on these observations, we hypothesize that progesterone induces reactivation in a subset of calves latently infected with BoHV-1. These studies demonstrated progesterone was less efficient than dexamethasone at initiating reactivation from latency in female calves. Notably, heat stress correlated with enhancing the ability of progesterone to induce reactivation from latency. Previous studies demonstrated that heat stress activates the glucocorticoid receptor (GR), which suggested GR activation augments progesterone-mediated reactivation from latency. Additional studies revealed GR and PR cooperatively stimulated productive infection and synergistically transactivated the IEtu1 promoter when cultures were treated with dexamethasone. Mutating one or both GR binding sites in the IEtu1 promoter blocked transactivation. Collectively, these studies indicated that progesterone intermittently triggered reactivation from latency, and heat stress augmented reactivation from reactivation. Finally, these studies suggest progesterone enhances virus spread in tissues and cells where PR is abundantly expressed. IMPORTANCE Steroid hormone fluctuations are predicted to enhance or initiate bovine herpesvirus 1 (BoHV-1) replication and virus spread in cattle. For example, stress increases the incidence of BoHV-1 reactivation from latency in cattle, and the synthetic corticosteroid dexamethasone consistently induces reactivation from latency. The glucocorticoid receptor (GR) and dexamethasone stimulate key viral regulatory promoters and productive infection, in part because the viral genome contains numerous consensus GR-responsive elements (GREs). The progesterone receptor (PR) and GR belong to the type I nuclear hormone receptor family. PR and progesterone specifically bind to and transactivate viral promoters that contain GREs and stimulate BoHV-1 productive infection. Although progesterone did not induce reactivation from latency in female calves as efficiently as dexamethasone, heat stress enhanced progesterone-mediated reactivation from latency. Consequently, we predict that low levels of stressful stimuli can cooperate with progesterone to induce reactivation from latency or promote virus spread.