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Infection by Kaposi sarcoma-associated herpesvirus (KSHV) can cause severe consequences, such as cancers and lymphoproliferative diseases. Whole inactivated viruses (WIV) with chemically destroyed genetic materials have been used as antigens in several licensed vaccines. During KSHV productive replication, virus-like vesicles (VLVs) that lack capsids and viral genomes are generated along with virions. Here, we investigated the immunogenicity of KSHV VLVs produced from a viral mutant that was defective in capsid formation and DNA packaging. Mice immunized with adjuvanted VLVs generated KSHV-specific T cell and antibody responses. Neutralization of KSHV infection by the VLV immune serum was low but was markedly enhanced in the presence of the complement system. Complement-enhanced neutralization and complement deposition on KSHV-infected cells was dependent on antibodies targeting viral open reading frame 4 (ORF4). However, limited complement-mediated enhancement was detected in the sera of a small cohort of KSHV-infected humans which contained few neutralizing antibodies. Therefore, vaccination that induces antibody effector functions can potentially improve infection-induced humoral immunity. Overall, our study highlights a potential benefit of engaging complement-mediated antibody functions in future KSHV vaccine development. IMPORTANCE KSHV is a virus that can lead to cancer after infection. A vaccine that prevents KSHV infection or transmission would be helpful in preventing the development of these cancers. We investigated KSHV VLV as an immunogen for vaccination. We determined that antibodies targeting the viral protein ORF4 induced by VLV immunization could engage the complement system and neutralize viral infection. However, ORF4-specific antibodies were seldom detected in the sera of KSHV-infected humans. Moreover, these human sera did not potently trigger complement-mediated neutralization, indicating an improvement that immunization can confer. Our study s

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 bp resolution and constructed a 3D KSHV genomic structural model with 2 kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by structural maintenance of chromosomes (SMC1) cohesin complex and CCCTC-binding factor (CTCF), and the KSHV transactivator was recruited to those sites during reactivation. Triggering KSHV gene expression decreased prewired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible noncoding RNA regions moved toward the inner space of the structure, resembling the configuration of a "bird cage" during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help facilitate viral gene transcription. IMPORTANCE The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi's sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibit

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 bp resolution and constructed a 3D KSHV genomic structural model with 2 kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by structural maintenance of chromosomes (SMC1) cohesin complex and CCCTC-binding factor (CTCF), and the KSHV transactivator was recruited to those sites during reactivation. Triggering KSHV gene expression decreased prewired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible noncoding RNA regions moved toward the inner space of the structure, resembling the configuration of a "bird cage" during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help facilitate viral gene transcription. IMPORTANCE The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi's sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibit

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4+ Tfh cells), which help B cells produce antibodies, were compared between HSV-1-infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that (i) the ASYMP individuals had increased frequencies of HSV-specific CD19+CD27+ memory B cells, and (ii) high frequencies of HSV-specific switched IgG+CD19+CD27+ memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0+CXCR5+CD4+ memory Tfh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD+CD138+ B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blindin

Studies on "hit-and-run" effects by viral proteins are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled identification of both static and dynamic protein-protein interactions. In this study, we applied a PL method by generating recombinant Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV, a gammaherpesvirus, uniquely encodes four interferon regulatory factors (IRF-1 to -4) that suppress host interferon responses, and we examined KSHV IRF-1 and IRF-4 neighbor proteins to identify cellular proteins involved in innate immune regulation. PL identified 213 and 70 proteins as neighboring proteins of viral IRF-1 (vIRF-1) and vIRF-4 during viral reactivation, and 47 proteins were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological roles such as mRNA processing and transcriptional regulation by TP53. Innate immune regulation by these commonly interacting 44 cellular proteins was examined with small interfering RNAs (siRNAs), and the splicing factor 3B family proteins were found to be associated with interferon transcription and to act as suppressors of KSHV reactivation. We propose that recombinant mini-TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication and that selective splicing factors have a function in the regulation of innate immune responses.IMPORTANCE Viral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to the use of affinity precipitation approaches, the majority of studies

Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication.IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene

Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular patholog

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinas

Herpes simplex virus 2 (HSV-2) is a common sexually transmitted infection with a highly variable clinical course. Many infections quickly become subclinical, with episodes of spontaneous virus reactivation. To study host-HSV-2 interactions, an animal model of subclinical HSV-2 infection is needed. In an effort to develop a relevant model, rhesus macaques (RM) were inoculated intravaginally with two or three HSV-2 strains (186, 333, and/or G) at a total dose of 1 × 107 PFU of HSV-2 per animal. Infectious HSV-2 and HSV-2 DNA were consistently shed in vaginal swabs for the first 7 to 14 days after each inoculation. Proteins associated with wound healing, innate immunity, and inflammation were significantly increased in cervical secretions immediately after HSV-2 inoculation. There was histologic evidence of acute herpesvirus pathology, including acantholysis in the squamous epithelium and ballooning degeneration of and intranuclear inclusion bodies in epithelial cells, with HSV antigen in mucosal epithelial cells and keratinocytes. Further, an intense inflammatory infiltrate was found in the cervix and vulva. Evidence of latent infection and reactivation was demonstrated by the detection of spontaneous HSV-2 shedding post-acute inoculation (102 to 103 DNA copies/swab) in 80% of RM. Further, HSV-2 DNA was detected in ganglia in most necropsied animals. HSV-2-specifc T-cell responses were detected in all animals, although antibodies to HSV-2 were detected in only 30% of the animals. Thus, HSV-2 infection of RM recapitulates many of the key features of subclinical HSV-2 infection in women but seems to be more limited, as virus shedding was undetectable more than 40 days after the last virus inoculation.IMPORTANCE Herpes simplex virus 2 (HSV-2) infects nearly 500 million persons globally, with an estimated 21 million incident cases each year, making it one of the most common sexually transmitted infections (STIs). HSV-2 is associated with increased human immunodeficiency v

A large proportion of the world population harbors herpes simplex virus 1 (HSV-1), a major cause of infectious corneal blindness. HSV-specific CD8+ T cells protect from herpesvirus infection and disease. However, the genomic, phenotypic, and functional characteristics of CD8+ T cells associated with the protection seen in asymptomatic (ASYMP) individuals, who, despite being infected, never experienced any recurrent herpetic disease, remains to be fully elucidated. In this investigation, we compared the phenotype, function, and level of expression of a comprehensive panel of 579 immune genes of memory CD8+ T cells, sharing the same HSV-1 epitope specificities, and freshly isolated peripheral blood from well-characterized cohorts of protected ASYMP and nonprotected symptomatic (SYMP) individuals, with a history of numerous episodes of recurrent herpetic disease, using the high-throughput digital NanoString nCounter system and flow cytometry. Interestingly, our results demonstrated that memory CD8+ T cells from ASYMP individuals expressed a unique set of genes involved in expansion and survival, type I interferon (IFN-I), and JAK/STAT pathways. Frequent multifunctional HSV-specific effector memory CD62Llow CD44high CD8+ TEM cells were detected in ASYMP individuals compared to more of monofunctional central memory CD62Lhigh CD44high CD8+ TCM cells in SYMP individuals. Shedding light on the genotype, phenotype, and function of antiviral CD8+ T cells from "naturally protected" ASYMP individuals will help design future T-cell-based ocular herpes immunotherapeutic vaccines.IMPORTANCE A staggering number of the world population harbors herpes simplex virus 1 (HSV-1) potentially leading to blinding recurrent herpetic disease. While the majority are asymptomatic (ASYMP) individuals who never experienced any recurrent herpetic disease, symptomatic (SYMP) individuals have a history of numerous episodes of recurrent ocular herpetic disease. This study elucidates the phenotype, th

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

Recently, we reported that the herpesvirus entry mediator (HVEM; also called TNFRSF14 or CD270) is upregulated by the latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1) and that the absence of HVEM affects latency reactivation but not primary infection in ocularly infected mice. gD has been shown to bind to HVEM. LIGHT (TNFSF14), CD160, and BTLA (B- and T-lymphocyte attenuator) also interact with HVEM and can interfere with HSV gD binding. It was not known if LIGHT, CD160, or BTLA affected the level of latency reactivation in the trigeminal ganglia (TG) of latently infected mice. To address this issue, we ocularly infected LIGHT-/-, CD160-/-, and BTLA-/- mice with LAT(+) and LAT(-) viruses, using similarly infected wild-type (WT) and HVEM-/- mice as controls. The amount of latency, as determined by the levels of gB DNA in the TG of the LIGHT-/-, CD160-/-, and BTLA-/- mice infected with either LAT(+) or LAT(-) viruses, was lower than that in WT mice infected with LAT(+) virus and was similar in WT mice infected with LAT(-) virus. The levels of LAT RNA in HVEM-/-, LIGHT-/-, CD160-/-, and BTLA-/- mice infected with LAT(+) virus were similar and were lower than the levels of LAT RNA in WT mice. However, LIGHT-/-, CD160-/-, and BTLA-/- mice, independent of the presence of LAT, had levels of reactivation similar to those of WT mice infected with LAT(+) virus. Faster reactivation correlated with the upregulation of HVEM transcript. The LIGHT-/-, CD160-/-, and BTLA-/- mice had higher levels of HVEM expression, and this, along with the absence of BTLA, LIGHT, or CD160, may contribute to faster reactivation, while the absence of each molecule, independent of LAT, may have contributed to lower latency. This study suggests that, in the absence of competition with gD for binding to HVEM, LAT RNA is important for WT levels of latency but not for WT levels of reactivation.IMPORTANCE The effects of BTLA, LIGHT, and CD160 on latency reactivation are not known. We

UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results s

Subclinical viral infections (SVI), including cytomegalovirus (CMV), are highly prevalent in humans, resulting in lifelong persistence. However, the impact of SVI on the interplay between the host immunity and gut microbiota in the context of environmental exposures is not well defined. We utilized the preclinical nonhuman primate (NHP) model consisting of SVI-free (specific-pathogen-free [SPF]) rhesus macaques and compared them to the animals with SVI (non-SPF) acquired through natural exposure and investigated the impact of SVI on immune cell distribution and function, as well as on gut microbiota. These changes were examined in animals housed in the outdoor environment compared to the controlled indoor environment. We report that SVI are associated with altered immune cell subsets and gut microbiota composition in animals housed in the outdoor environment. Non-SPF animals harbored a higher proportion of potential butyrate-producing Firmicutes and higher numbers of lymphocytes, effector T cells, and cytokine-producing T cells. Surprisingly, these differences diminished following their transfer to the controlled indoor environment, suggesting that non-SPFs had increased responsiveness to environmental exposures. An experimental infection of indoor SPF animals with CMV resulted in an increased abundance of butyrate-producing bacteria, validating that CMV enhanced colonization of butyrate-producing commensals. Finally, non-SPF animals displayed lower antibody responses to influenza vaccination compared to SPF animals. Our data show that subclinical CMV infection heightens host immunity and gut microbiota changes in response to environmental exposures. This may contribute to the heterogeneity in host immune response to vaccines and environmental stimuli at the population level.IMPORTANCE Humans harbor several latent viruses that modulate host immunity and commensal microbiota, thus introducing heterogeneity in their responses to pathogens, vaccines, and environmental

Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these

Bivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression, while the H3K4me3 mark prevents the region from becoming permanently silenced and prepares the domain for activation when needed. Specific regions of Kaposi's sarcoma-associated herpesvirus (KSHV) latent episomes are poised to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function; thus, we hypothesized that identification of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication. Here we show that a zinc finger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquitinate ZIC2. ZIC2 localized at immediate early and early gene cluster regions of the KSHV genome and contributed to tethering of polycomb repressive complex 2 through physical interaction, thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted the balance of bivalent histone modifications toward more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication.IMPORTANCE Posttranslational histone modifications regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reactivatable by K-Rta remains uncle

There are no approved therapeutics for the treatment of dengue disease despite the global prevalence of dengue virus (DENV) and its mosquito vectors. DENV infections can lead to vascular complications, hemorrhage, and shock due to the ability of DENV to infect a variety of immune and nonimmune cell populations. Increasingly, studies have implicated the host response as a major contributor to severe disease. Inflammatory products of various cell types, including responding T cells, mast cells (MCs), and infected monocytes, can contribute to immune pathology. In this study, we show that the host response to DENV infection in immunocompetent mice recapitulates transcriptional changes that have been described in human studies. We found that DENV infection strongly induced metabolic dysregulation, complement signaling, and inflammation. DENV also affected the immune cell content of the spleen and liver, enhancing NK, NKT, and CD8+ T cell activation. The MC-stabilizing drug ketotifen reversed many of these responses without suppressing memory T cell formation and induced additional changes in the transcriptome and immune cell composition of the spleen, consistent with reduced inflammation. This study provides a global transcriptional map of immune activation in DENV target organs of an immunocompetent host and supports the further development of targeted immunomodulatory strategies to treat DENV disease.IMPORTANCE Dengue virus (DENV), which causes febrile illness, is transmitted by mosquito vectors throughout tropical and subtropical regions of the world. Symptoms of DENV infection involve damage to blood vessels and, in rare cases, hemorrhage and shock. Currently, there are no targeted therapies to treat DENV infection, but it is thought that drugs that target the host immune response may be effective in limiting symptoms that result from excessive inflammation. In this study, we measured the host transcriptional response to infection in multiple DENV target organs using

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107a/b cytotoxic degranulation. High frequencies of multifunctional CD8+ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286-294), VP13/14 from amino acids 504 to 512 (VP13/14504-512), and VP13/14 from amino acids 544 to 552 (VP13/14544-552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RAlow CD44high CCR7low CD62Llow CD8+ effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion ly

Virus-like vesicles (VLVs) are membrane-enclosed vesicles that resemble native enveloped viruses in organization but lack the viral capsid and genome. During the productive infection of tumor-associated gammaherpesviruses, both virions and VLVs are produced and are released into the extracellular space. However, studies of gammaherpesvirus-associated VLVs have been largely restricted by the technical difficulty of separating VLVs from mature virions. Here we report a strategy of selectively isolating VLVs by using a Kaposi's sarcoma-associated herpesvirus (KSHV) mutant that is defective in small capsid protein and is unable to produce mature virions. Using mass spectrometry analysis, we found that VLVs contained viral glycoproteins required for cellular entry, as well as tegument proteins involved in regulating lytic replication, but lacked capsid proteins. Functional analysis showed that VLVs induced the expression of the viral lytic activator RTA, initiating KSHV lytic gene expression. Furthermore, employing RNA sequencing, we performed a genomewide analysis of cellular responses triggered by VLVs and found that PRDM1, a master regulator in cell differentiation, was significantly upregulated. In the context of KSHV replication, we demonstrated that VLV-induced upregulation of PRDM1 was necessary and sufficient to reactivate KSHV by activating its RTA promoter. In sum, our study systematically examined the composition of VLVs and demonstrated their biological roles in manipulating host cell responses and facilitating KSHV lytic replication.IMPORTANCE Cells lytically infected with tumor-associated herpesviruses produce a high proportion of virus-like vesicles (VLVs). The composition and function of VLVs have not been well defined, largely due to the inability to efficiently isolate VLVs that are free of virions. Using a cell system capable of establishing latent KSHV infection and robust reactivation, we successfully isolated VLVs from a KSHV mutant defective in the

Despite the high incidence of severe defects in the central nervous system caused by human cytomegalovirus (HCMV) congenital infection, the mechanism of HCMV neuropathogenesis and the roles of individual viral genes have not yet been fully determined. In this study, we show that the immediate-early 2 (IE2) protein may play a key role in HCMV-caused neurodevelopmental disorders. IE2-transduced neural progenitor cells gave rise to neurospheres with a lower frequency and produced smaller neurospheres than control cells in vitro, indicating reduction of self-renewal and expansion of neural progenitors by IE2. At 2 days after in utero electroporation into the ventricle of the developing brain, a dramatically lower percentage of IE2-expressing cells was detected in the ventricular zone (VZ) and cortical plate (CP) compared to control cells, suggesting that IE2 concurrently dysregulates neural stem cell maintenance in the VZ and neuronal migration to the CP. In addition, most IE2+ cells in the lower intermediate zone either showed multipolar morphology with short neurites or possessed nonradially oriented processes, whereas control cells had long, radially oriented monopolar or bipolar neurites. IE2+ callosal axons also failed to cross the midline to form the corpus callosum. Furthermore, we provide molecular evidence that the cell cycle arrest and DNA binding activities of IE2 appear to be responsible for the increased neural stem cell exit from the VZ and cortical migrational defects, respectively. Collectively, our results demonstrate that IE2 disrupts the orderly process of brain development in a stepwise manner to further our understanding of neurodevelopmental HCMV pathogenesis.IMPORTANCE HCMV brain pathogenesis has been studied in limited experimental settings, such as in vitro HCMV infection of neural progenitor cells or in vivo murine CMV infection of the mouse brain. Here, we show that IE2 is a pivotal factor that contributes to HCMV-induced abnormalities in the