Kaposi sarcoma is a frequently aggressive malignancy caused by Kaposi sarcoma herpesvirus. People with immunodeficiencies, including human immunodeficiency virus (HIV), are at increased risk for developing Kaposi sarcoma, but our understanding of the contributions of the cellular genome to Kaposi sarcoma pathogenesis remains limited. To determine if there are cellular genetic alterations in Kaposi sarcoma that might provide biological or therapeutic insights, we performed whole-exome sequencing on 78 Kaposi sarcoma tumors and matched normal control skin from 59 adults with Kaposi sarcoma (46 with HIV-associated Kaposi sarcoma and 13 with HIV-negative Kaposi sarcoma) receiving treatment at the Uganda Cancer Institute in Kampala, Uganda. We found a very low mutational burden in all but one specimen (median = 11 mutations), which is the lowest number of mutations among all 33 tumor types in The Cancer Genome Atlas. No recurrent mutations were seen, and the most commonly affected oncogenic pathway was RTK/RAS. Mutational signatures included defective DNA mismatch repair and smoking. There was no evidence suggesting that multiple tumors from the same patient originated from the same original clone. The number of genome copy alterations per genome was higher in tumors from those without HIV infection and in tumors from participants with advanced stage disease, suggesting that lesions that take longer to develop may accumulate more alterations, although the number of alterations remains low compared with other cancers. Implications: Our findings indicate that the pathogenesis of Kaposi sarcoma differs from other malignancies and that the primary driver of carcinogenesis is Kaposi sarcoma-associated herpesvirus infection and expression of viral oncogenes, rather than clonal oncogenic transformation.

Kaposi's sarcoma (KS) is a vascular intermediate malignant tumor classified into four clinical types: classic, AIDS-related, iatrogenic, and endemic. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of KS. Six KSHV genotypes (A, B, C, D, E, and F) classified by K1 or two genotypes (P and M) by K15 have been reported. However, whether the KSHV genotype affects clinical presentation remains elusive. Herein, we investigated the association between viral genotypes and clinical presentations in patients with KS in Okinawa, an endemic area in Japan. Classic KS caused by KSHV genotype C was identified as the most common clinical type of KS in Okinawa. Conversely, 80% of the patients with AIDS-related KS were associated with genotype A. According to K15 genotyping, the population of genotype M was higher than that of genotype P. Although genotype M accounted for most cases of both classic and iatrogenic KS in Okinawa, genotype P constituted the majority of AIDS-related KS. Regarding the association between the K1 and K15 genotypes, single genotype A was associated with genotype P, whereas single genotype C was associated with genotype M. These K1 and K15 associations in Okinawa differed from those in Europe and Africa. In terms of the association between viral genotype and clinical types, A/P tended to be associated with AIDS-related KS and genotype C/M tended to be associated with classic KS. The findings of the current study suggest that the KSHV genotype in Okinawa differs from that in other countries, which is related to the KSHV geographic distribution and population migration. Our data also suggest that the viral genotype in Okinawa is associated with clinical presentations.

Kaposi sarcoma (KS) is the most common cancer in people living with HIV (PLWH), particularly in sub-Saharan Africa (SSA), where Kaposi sarcoma herpesvirus (KSHV or human herpesvirus 8 [HHV-8]) is endemic. In KSHV endemic areas, the overall survival of KS patients has changed little over the past 20 years. A phylogenetic analysis of available full-length viral genomes (n = 164) identified two different virus lineages that co-circulate in KSHV endemic regions today. Their sequences differ from the GenBank reference sequence and those of common laboratory strains, which originated in the 1990s in the US and Europe. Targeted short-read sequencing accuracy was validated by PacBio-based long-read sequencing to resolve repeats. This analysis identified over 1,000 single nucleotide variants (SNV) in a new 14-member sequence collection from tumor biopsies and blood in Malawi with 127 ± 32 (median ± SD) SNV per genome. Most were private, i.e., specific to one individual's virus. Within each of the two lineages, KSHV continues to evolve over time and across national borders by genetic drift and recombination. Analyses of shared SNVs by AlphaFold2 predicted some changes in the conformation of key viral proteins. These findings may help our understanding of herpesvirus evolution.

To understand viruses, the field needs to know their genetic makeup. To develop mechanistic models, targeted therapies, and vaccines, we need comprehensive and up-to-date sequence information on the viral strains that circulate where the diseases appear today. Our knowledge of Kaposi sarcoma herpesvirus (KSHV) sequence distribution and evolution is behind that of other human herpesviruses and RNA viruses. Here, we add to community knowledge using new technologies and artificial intelligence.

Kaposi Sarcoma (KS) is a complex tumor caused by KS-associated herpesvirus 8 (KSHV). Histological analysis reveals a mixture of "spindle cells", vascular-like spaces, extravasated erythrocytes, and immune cells. In order to elucidate the infected and uninfected cell types in KS tumors, we examined twenty-five skin and blood samples from sixteen subjects by single cell RNA sequence analyses. Two populations of KSHV-infected cells were identified, one of which represented a CD34-negative proliferative fraction of endothelial cells, and the second representing CD34-positive cells expressing endothelial genes found in a variety of cell types including high endothelial venules, fenestrated capillaries, and endothelial tip cells. Although both infected clusters contained cells expressing lytic and latent KSHV genes, the CD34+ cells expressed more K5 and less K12. Novel cellular biomarkers were identified in the KSHV infected cells, including the sodium channel SCN9A. The number of KSHV positive cells was found to be less than 10% of total tumor cells in all samples and correlated inversely with tumor-infiltrating immune cells. T-cell receptor clones were expanded in KS tumors and blood, although in differing magnitudes. Changes in cellular composition in KS tumors after treatment with antiretroviral therapy alone, or immunotherapy were noted. These studies demonstrate the feasibility of single cell analyses to identify prognostic and predictive biomarkers.

With the widespread prevalence of Kaposi's sarcoma-associated herpesvirus (KSHV) globally, particularly in sub-Saharan Africa and the Mediterranean region, the open reading frame (ORF) K1 gene, a key gene for distinguishing different subtypes of KSHV, has been extensively studied for its diversity and sequence variations. In this study, we collected K1 gene sequences representing subtypes of KSHV worldwide in order to assess the global distribution of KSHV subtypes and to investigate the recombination and selection history of KSHV. Recombination and gene flow analysis indicated a minimum average recombination rate of 0.41 per site for the K1 gene. Recombination analysis indicated that 11 major recombination events had occurred, predominantly in subtypes A and C, while subtype B showed minimal involvement in recombination processes, consistent with the gene flow analysis. Using tip-dating methods, we estimated that the most recent common ancestor of KSHV emerged in the 12th century, while the currently globally prevalent subtypes appeared within the past three centuries. Its recent origin and rapid evolution indicate that KSHV is now undergoing strong selection and is in the process of adaptation.

Kaposi's sarcoma-associated herpesvirus (KSHV) genome contains a terminal repeats (TR) sequence. Previous studies demonstrated that KSHV TR functions as a gene enhancer for inducible lytic gene promoters. Gene enhancers anchor bromodomain-containing protein 4 (BRD4) at specific genomic region, where BRD4 interacts flexibly with transcription-related proteins through its intrinsically disordered domain and exerts transcription regulatory function. Here, we generated recombinant KSHV with reduced TR copy numbers and studied BRD4 recruitment and its contributions to the inducible promoter activation. Reducing the TR copy numbers from 21 (TR21) to 5 (TR5) strongly attenuated viral gene expression during de novo infection and impaired reactivation. The EF1α promoter encoded in the KSHV BAC backbone also showed reduced promoter activity, suggesting a global attenuation of transcription activity within TR5 latent episomes. Isolation of reactivating cells confirmed that the reduced inducible gene transcription from TR-shortened DNA template and is mediated by decreased efficacies of BRD4 recruitment to viral gene promoters. Separating the reactivating iSLK cell population from non-responders showed that reactivatable iSLK cells harbored larger LANA nuclear bodies (NBs) compared to non-responders. The cells with larger LANA NBs, either due to prior transcription activation or TR copy number, supported KSHV reactivation more efficiently than those with smaller LANA NBs. With auxin-inducible LANA degradation, we confirmed that LANA is responsible for BRD4 occupancies on latent chromatin. Finally, with purified fluorescence-tagged proteins, we demonstrated that BRD4 is required for LANA to form liquid-liquid phase-separated dots. The inclusion of TR DNA fragments further facilitated the formation of larger BRD4-containing LLPS in the presence of LANA, similar to the "cellular enhancer dot" formed by transcription factor-DNA bindings. These results suggest that LANA binding to TR establishes an enhancer domain for infected KSHV episomes. The strength of this enhancer, regulated by TR length or transcription memory, determines the outcome of KSHV replication.

The spindle cells of Kaposi sarcoma (KS) lesions primarily express Kaposi sarcoma herpesvirus (KSHV) latent genes with minimal expression of lytic genes. However, recent transcriptome analyses of KS lesions have shown high expression of KSHV open reading frame (ORF) 75, which is considered a late lytic gene based on analyses in primary effusion lymphoma (PEL) lines. ORF75 encodes a pseudo-amidotransferase that is part of the viral tegument, acts as a suppressor of innate immunity, and is essential for viral lytic replication. We assessed a representative KS lesion by RNAscope and found that ORF75 RNA was expressed in the majority of latency-associated nuclear antigen (LANA)-expressing cells. Luciferase fusion reporter constructs of the ORF75 promoter were analyzed for factors potentially driving its expression in KS. The ORF75 promoter construct showed high basal transcriptional activity in vitro in endothelial cells, mediated by a proximal consensus specificity protein 1 (Sp1) (GGGGCGGGGC) element along with two distal CCAAT boxes. Sp proteins formed complexes with the proximal consensus Sp1 element to activate ORF75 promoter transcription. We also found evidence that a repressive factor or factors in B cells, but not endothelial or epithelial cells, interacted with more distal elements in the ORF75 promoter region to repress constitutive ORF75 expression in B cells. Alternate forms of Sp1 were found to accumulate during latency and showed substantial enrichment during viral lytic replication in PEL cells and infected endothelial cells, but their functional significance is unclear. We also found that ORF75 can in turn upregulate its own expression and that of other KSHV genes. Thus, while ORF75 acts primarily as a lytic gene in PEL cell lines, Sp proteins induce substantial constitutive ORF75 transcription in infected endothelial cells and this can account for its high expression in KS lesions.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus belonging to the γ-herpesvirus subfamily. KSHV is the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman's disease (MCD), and KSHV inflammatory cytokine syndrome (KICS). Since its discovery, research on KSHV has rapidly progressed, but existing information platforms relatively lack comprehensiveness and do not provide efficient analysis tools tailored for KSHV. To further promote the research on KSHV more effectively, we have developed KSHVbook (http://www.kshvbook.com), a specialized information-sharing database dedicated to KSHV. This platform offers extensive information on genes, coding sequences, proteins, and the gene regulatory region. Besides, the KSHVbook includes about 35 010 transcription factor binding sites (TFBSs), 342 010 pairs of KSHV miRNA-host target gene relationships, protein structures predicted by AlphaFold3, qPCR primers, and so on. We also develop analytical tools for viral genome regions, TFBSs, and KSHV miRNA target genes to discover previously unknown biological functions of KSHV. These analytical tools can effectively identify the potential regulatory relationships between host transcription factors and viral genes. Overall, this platform provides a centralized data resource for KSHV research by integrating multiple databases, offering accessible analysis tools, and simplifying data acquisition. The KSHVbook will continue to be updated, and more features can be found on the website.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus associated with Kaposi's sarcoma and B cell malignancies. Like all herpesviruses, KSHV contains conserved envelope glycoproteins (gps) involved in virus binding, entry, assembly, and release from infected cells, which are also targets of the immune response. Due to the lack of a reproducible animal model of KSHV infection, the precise functions of the KSHV gps during infection in vivo are not completely known. Fortunately, a nonhuman primate (NHP) model of KSHV infection and disease has been established utilizing closely related rhesus macaque rhadinovirus (RRV) that naturally infects rhesus macaques (RM) and possesses analogous gps to KSHV. To address the roles conserved envelope gps gH and gL play during KSHV infection in vivo, we utilized the pathogenic RRV17577 BAC to generate chimeric forms of RRV expressing KSHV gL or KSHV gH/gL, as well as an RRV mutant lacking gL expression. These viruses incorporate KSHV gH and gL into infectious virions, and although they display variable replication and differing plaque phenotypes in primary rhesus fibroblasts, they retain the ability to infect human B cells in vitro. Importantly, we also demonstrate that RRV gp chimeras can infect RM and induce the development of antibodies against KSHV. Overall, this work demonstrates that RRV gp chimeras can serve as important tools to assess the role of KSHV gH/gL in infection and disease while also providing an NHP model for testing the efficacy of KSHV gH and gL neutralizing antibodies and vaccine strategies to prevent and treat KSHV infection.IMPORTANCERhesus macaque rhadinovirus (RRV) is a rhesus macaque homolog of KSHV and serves as a model system for examining Kaposi's sarcoma-associated herpesvirus (KSHV) infection and pathogenesis in vivo. KSHV and RRV both encode conserved herpesvirus envelope glycoproteins, including gH and gL, that are important for regulating entry into host cells. In this study, we utilized the RRV BAC system to generate chimeric forms of RRV expressing KSHV gH and gL, as well as a mutant form of RRV lacking gL expression. Although these mutant and chimeric viruses can replicate in vitro, they do display growth properties different from wild-type RRV. Importantly, we demonstrate that RRV gp chimeras are capable of infecting rhesus macaques in vivo, inducing B cell hyperplasia, and promoting the development of anti-viral antibody responses that can also recognize KSHV antigens. RRV gp chimeras provide a novel system that allows for the examination of the role of KSHV gH and gL during infection in vivo.

During viral infection, the transport of various proteins between the nucleus and cytoplasm plays an important role in the viral lifecycle. Shuttling proteins are key factors in the transmission of nucleocytoplasmic information within cells and usually contain nuclear localization signals and nuclear export signals to mediate correct positioning for themselves and other proteins. The nucleocytoplasmic transport process is carried out through the nuclear pore complex on the nuclear envelope and is mediated by specific protein carriers. The viral proteins that function through nucleocytoplasmic shuttling in herpesviruses have gradually been identified as research advances. This article provides an overview of how shuttling proteins utilize nucleocytoplasmic shuttling signals and nuclear transport receptors for nucleocytoplasmic transport, as well as discusses how herpesvirus shuttling proteins enhance the effective infection of viruses by affecting their lifecycle and participating in innate immunity, this review provides a reference for understanding the pathogenesis of herpesvirus infection and determining new antiviral strategies.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and the etiological agent of several diseases. These include the malignancies Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), as well as the inflammatory disorder KSHV inflammatory cytokine syndrome (KICS). The KSHV lifecycle is characterized by two phases: a default latent phase and a lytic replication cycle. During latency, the virus persists as an episome within host cells, expressing a limited subset of viral genes to evade immune surveillance while promoting cellular transformation. The lytic phase, triggered by various stimuli, results in the expression of the full viral genome, production of infectious virions, and modulation of the tumor microenvironment. Both phases of the KSHV lifecycle play crucial roles in driving viral pathogenesis, influencing oncogenesis and immune evasion. This review dives into the intricate world of the KSHV lifecycle, focusing on the molecular mechanisms that drive its latent and lytic phases, their roles in disease progression, and current therapeutic strategies.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA gamma herpesvirus. Like other herpesviruses, KSHV establishes a latent infection with limited gene expression, while KSHV occasionally undergoes the lytic replication phase, which produces KSHV progenies and infects neighboring cells. KSHV genome encodes 80+ open reading frames. One of the KSHV genes, K2, encodes viral interleukin 6 (vIL-6), a homolog of human IL-6 (hIL-6), mainly expressed in the lytic phase of the virus. vIL-6 plays a crucial role in regulating the expression of other viral genes and is also associated with inducing angiogenesis, cell survival, and immune evasion, which is suggested to promote the development of KSHV-associated diseases. This review summarizes the current knowledge on vIL-6. We focus on the vIL-6 regarding its protein structure, transcriptional regulation, cell signaling pathways, and contribution to the KSHV-associated diseases.

"When you can measure what you are speaking about, and express it in numbers, you know something about it." is a famous quote attributed to Lord Kelvin. This sentiment puts viral load measurements at the center of virology. Viral load, or more precisely, DNA copy number measurements, are also used to follow infections with human herpesviruses, such as Kaposi sarcoma herpesvirus (KSHV) and Epstein-Barr Virus (EBV). EBV and KSHV are associated with human cancers, and determining their DNA copy numbers in the context of cancer prediction and progression on therapy is of fundamental scientific and translational interest. Yet, there is no generally accepted assay for KSHV DNA quantitation, and KSHV viral load is not used in clinical decision-making. Here, we review the history of KSHV DNA detection assays, explore factors that affect sensitivity and specificity, and describe an automated, high-throughput, real-time quantitative polymerase chain reaction (PCR) assay for KSHV and EBV. In conjunction with a digital PCR assay using the same primer/probe combination, we describe how to determine the absolute KSHV genome copy numbers in plasma, peripheral blood mononuclear cells, saliva, and other easily accessible body fluids.

Kaposi's sarcoma-associated herpesvirus (KSHV), a γ-herpesvirus, is predominantly associated with Kaposi's sarcoma (KS) as well as two lymphoproliferative disorders: primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other herpesviruses, KSHV employs two distinct life cycles: latency and lytic replication. To establish a lifelong persistent infection, KSHV has evolved various strategies to manipulate the epigenetic machinery of the host. In latently infected cells, most viral genes are epigenetically silenced by components of cellular chromatin, DNA methylation and histone post-translational modifications. However, some specific latent genes are preserved and actively expressed to maintain the virus's latent state within the host cell. Latency is not a dead end, but the virus has the ability to reactivate. This reactivation is a complex process that involves the removal of repressive chromatin modifications and increased accessibility for both viral and cellular factors, allowing the activation of the full transcriptional program necessary for the subsequent lytic replication. This review will introduce the roles of epigenetic modifications in KSHV latent and lytic life cycles, including DNA methylation, histone methylation and acetylation modifications, chromatin remodeling, genome conformation, and non-coding RNA expression. Additionally, we will also review the transcriptional regulation of viral genes and host factors in KSHV infection. This review aims to enhance our understanding of the molecular mechanisms of epigenetic modifications and transcriptional regulation in the KSHV life cycle, providing insights for future research.

Approximately 15-20% of cancers are caused by viruses. Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is an oncogenic virus that is the etiologic agent of not only Kaposi sarcoma but also the lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). KSHV can infect a broad tropism of cells, including B lymphocytes, wherein KSHV encodes specific viral proteins that can transform the cell. KSHV infection precedes the progression of PEL and MCD. KSHV establishes lifelong infection and has two phases of its lifecycle: latent and lytic. During the latent phase, viral genomes are maintained episomally with limited gene expression. Upon sporadic reactivation, the virus enters its replicative lytic phase to produce infectious virions. KSHV relies on its viral products to modulate host factors to evade immune detection or to co-opt their function for KSHV persistence. These manipulations dysregulate normal cell pathways to ensure cell survival and inhibit antiviral immune responses, which in turn, contribute to KSHV-associated malignancies. Here, we highlight the known molecular mechanisms of KSHV that promote lymphomagenesis and how these findings identify potential therapeutic targets for KSHV-associated lymphomas.

Herpesviruses are a family of double-stranded DNA viruses with a tegument structure and a genome composed of a single sequence and terminal repeat (TR) sequences. The herpesvirus UL14 gene encodes the protein UL14 (pUL14), which has various subcellular localizations and plays a vital role in regulating immediate-early (IE) gene transcription and expression, influences the intracellular localization patterns of several proteins belonging to the capsid and the DNA packaging machinery, participates in secondary envelopment, and influences viral particle release. Additionally, pUL14 has roles in maintaining cellular homeostasis and preventing apoptosis. This review discusses how pUL14 engages in the life cycle of herpesviruses and provides new ideas for further research on pUL14's function in viral infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus that establishes life-long infection. KSHV is implicated in the etiology of Kaposi's sarcoma, and a number of rare hematopoietic malignancies. The present study focuses on the KSHV open reading frame 20 (ORF20), a member of the conserved herpesvirus UL24 protein family containing five conserved homology domains and a conserved PD-(D/E)XK putative endonuclease motif, whose nuclease function has not been established to date. ORF20 encodes three co-linear protein isoforms, full length, intermediate, and short, though their differential functions are unknown. In an effort to determine the role of ORF20 during KSHV infection, we generated a recombinant ORF20-Null KSHV genome, which fails to express all three ORF20 isoforms. This genome was reconstituted in iSLK cells to establish a latent infection, which resulted in an accelerated transcription of viral mRNAs, an earlier accumulation of viral lytic proteins, an increase in the quantity of viral DNA copies, and a significant decrease in viral yield upon lytic reactivation. This was accompanied by early cell death of cells infected with the ORF20-Null virus. Functional complementation of the ORF20-Null mutant with the short ORF20 isoform rescued KSHV production, whereas its endonuclease mutant form failed to enhance lytic reactivation. Complementation with the short isoform further revealed a decrease in cell death as compared with ORF20-Null virus. Finally, expression of IL6 and CXCL8, previously shown to be affected by the hCMV UL24 homolog, was relatively low upon reactivation of cells infected with the ORF20-Null virus. These findings suggest that ORF20 protein, with its putative endonuclease motif, promotes coordinated lytic reactivation for increased infectious particle production.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus with a double-stranded DNA capable of establishing latent infection in the host cell. During latency, only a limited number of viral genes are expressed in infected host cells, and that helps the virus to evade host immune cell response. During primary infection, the KSHV genome is chromatinized and maintained as an episome, which is tethered to the host chromosome via Latency Associated Nuclear Antigen (LANA). The KSHV episome undergoes the same chromatin modification with the host cell chromosome and, therefore, is regulated by various epigenetic modifications, such as DNA methylation, histone methylation, and histone acetylation. The KSHV genome is also organized in a spatiotemporal manner by forming genomic loops, which enable simultaneous and coordinated control of dynamic gene transcription, particularly during the lytic replication phase. The genome-wide approaches and advancing bioinformatic tools have increased the resolution of studies on the dynamic transcriptional control and our understanding of KSHV latency-lytic switch regulation. We will summarize our current understanding of the epigenetic gene regulation on the KSHV chromatin.

Of the thousands of viruses infecting humans, only seven cause cancer in the general population. Tumor sequencing is now a common cancer medicine procedure, and so it seems likely that more human cancer viruses already would have been found if they exist. Here, we review cancer characteristics that can inform a dedicated search for new cancer viruses, focusing on Kaposi sarcoma herpesvirus and Merkel cell polyomavirus as the most recent examples of successful genomic and transcriptomic searches. We emphasize the importance of epidemiology in determining which cancers to examine and describe approaches to virus discovery. Barriers to virus discovery, such as novel genomes and viral suppression of messenger RNA expression, may exist that prevent virus discovery using existing approaches. Optimally virus hunting should be performed in such a way that if no virus is found, the tumor can be reasonably excluded from having an infectious etiology and new information about the biology of the tumor can be found.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Kaposi Sarcoma (KS) is a complex tumor caused by KS-associated herpesvirus 8 (KSHV). Histological analysis reveals a mixture of "spindle cells", vascular-like spaces, extravasated erythrocytes, and immune cells. In order to elucidate the infected and uninfected cell types in KS tumors, we examined skin and blood samples from twelve subjects by single cell RNA sequence analyses. Two populations of KSHV-infected cells were identified, one of which represented a proliferative fraction of lymphatic endothelial cells, and the second represented an angiogenic population of vascular endothelial tip cells. Both infected clusters contained cells expressing lytic and latent KSHV genes. Novel cellular biomarkers were identified in the KSHV infected cells, including the sodium channel SCN9A. The number of KSHV positive tumor cells was found to be in the 6% range in HIV-associated KS, correlated inversely with tumor-infiltrating immune cells, and was reduced in biopsies from HIV-negative individuals. T-cell receptor clones were expanded in KS tumors and blood, although in differing magnitudes. Changes in cellular composition in KS tumors were identified in subjects treated with antiretroviral therapy alone, or immunotherapy. These studies demonstrate the feasibility of single cell analyses to identify prognostic and predictive biomarkers.

Kaposi sarcoma (KS) is a malignancy caused by the KS-associated herpesvirus (KSHV) that causes skin lesions, and may also be found in lymph nodes, lungs, gastrointestinal tract, and other organs in immunosuppressed individuals more commonly than immunocompetent subjects. The current study examined gene expression in single cells from the tumor and blood of these subjects, and identified the characteristics of the complex mixtures of cells in the tumor. This method also identified differences in KSHV gene expression in different cell types and associated cellular genes expressed in KSHV infected cells. In addition, changes in the cellular composition could be elucidated with therapeutic interventions.

Septin proteins are a subfamily of closely related GTP-binding proteins conserved in all species except for higher plants and perform essential biological processes. Septins self-assemble into heptameric or octameric complexes and form higher-order structures such as filaments, rings, or gauzes by end-to-end binding. Their close association with cell membrane components makes them central in regulating critical cellular processes. Due to their organisation and properties, septins function as diffusion barriers and are integral in providing scaffolding to support the membrane's curvature and stability of its components. Septins are also involved in vesicle transport and exocytosis through the plasma membrane by co-localising with exocyst protein complexes. Recently, there have been emerging reports of several human and animal diseases linked to septins and abnormalities in their functions. Most of our understanding of the significance of septins during microbial diseases mainly pertains to their roles in bacterial infections but not viruses. This present review focuses on the known roles of septins in host-viral interactions as detailed by various studies.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

Herpesviruses are large DNA viruses and include important human and veterinary pathogens. Their genomes can be cloned as bacterial artificial chromosomes (BACs) and genetically engineered in Escherichia coli using BAC recombineering methods. While the recombineering methods are efficient, the initial BAC-cloning step remains laborious. To overcome this limitation, we have developed a simple, rapid, and efficient BAC-cloning method based on single-step transformation-associated recombination (STAR) in Saccharomyces cerevisiae. The linear viral genome is directly integrated into a vector comprising a yeast centromeric plasmid and a BAC replicon. Following transfer into E. coli, the viral genome can be modified using standard BAC recombineering techniques. We demonstrate the speed, fidelity, and broad applicability of STAR by cloning two strains of both rat cytomegalovirus (a betaherpesvirus) and Kaposi's sarcoma-associated herpesvirus (a gammaherpesvirus). STAR cloning facilitates the functional genetic analysis of herpesviruses and other large DNA viruses and their use as vaccines and therapeutic vectors.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

MCL-1 is the prosurvival member of the Bcl-2 family. It prevents the induction of mitochondria-dependent apoptosis. The molecular mechanisms dictating the host cell viability gain importance in the context of viral infections. The premature apoptosis of infected cells could interrupt the pathogen replication cycle. On the other hand, cell death following the effective assembly of progeny particles may facilitate virus dissemination. Thus, various viruses can interfere with the apoptosis regulation network to their advantage. Research has shown that viral infections affect the intracellular amount of MCL-1 to modify the apoptotic potential of infected cells, fitting it to the "schedule" of the replication cycle. A growing body of evidence suggests that the virus-dependent deregulation of the MCL-1 level may contribute to several virus-driven diseases. In this work, we have described the role of MCL-1 in infections caused by various viruses. We have also presented a list of promising antiviral agents targeting the MCL-1 protein. The discussed results indicate targeted interventions addressing anti-apoptotic MCL1 as a new therapeutic strategy for cancers as well as other diseases. The investigation of the cellular and molecular mechanisms involved in viral infections engaging MCL1 may contribute to a better understanding of the regulation of cell death and survival balance.

Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell's DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi's sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.

The phylum Nucleocytoviricota includes the largest and most complex viruses known. These "giant viruses" have a long evolutionary history that dates back to the early diversification of eukaryotes, and over time they have evolved elaborate strategies for manipulating the physiology of their hosts during infection. One of the most captivating of these mechanisms involves the use of genes acquired from the host-referred to here as viral homologs or "virologs"-as a means of promoting viral propagation. The best-known examples of these are involved in mimicry, in which viral machinery "imitates" immunomodulatory elements in the vertebrate defense system. But recent findings have highlighted a vast and rapidly expanding array of other virologs that include many genes not typically found in viruses, such as those involved in translation, central carbon metabolism, cytoskeletal structure, nutrient transport, vesicular trafficking, and light harvesting. Unraveling the roles of virologs during infection as well as the evolutionary pathways through which complex functional repertoires are acquired by viruses are important frontiers at the forefront of giant virus research.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA (dsDNA) gammaherpesvirus with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5; however, ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus and cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production. IMPORTANCE Although the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the Kaposi's sarcoma-associated herpesvirus (KSHV) terminase complex is still largely uncharacterized. This complex presumably contains KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for terminal repeat (TR) cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the artificial intelligence (AI)-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.

Kaposi sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8, is an oncogenic herpesvirus. Its latency-associated nuclear antigen (LANA) is essential for the persistence of KSHV in latently infected cells. LANA mediates replication of the latent viral genome during the S phase of a dividing cell and partitions episomes to daughter cells by attaching them to mitotic chromosomes. It also mediates the establishment of latency in newly infected cells through epigenetic mechanisms and suppresses the activation of the productive replication cycle. Furthermore, LANA promotes the proliferation of infected cell by acting as a transcriptional regulator and by modulating the cellular proteome through the recruitment of several cellular ubiquitin ligases. Finally, LANA interferes with the innate and adaptive immune system to facilitate the immune escape of infected cells.

Epstein-Barr virus (EBV) infects host cells and establishes a latent infection that requires evasion of host innate immunity. A variety of EBV-encoded proteins that manipulate the innate immune system have been reported, but whether other EBV proteins participate in this process is unclear. EBV-encoded envelope glycoprotein gp110 is a late protein involved in virus entry into target cells and enhancement of infectivity. Here, we reported that gp110 inhibits RIG-I-like receptor pathway-mediated promoter activity of interferon-β (IFN-β) as well as the transcription of downstream antiviral genes to promote viral proliferation. Mechanistically, gp110 interacts with the inhibitor of NF-κB kinase (IKKi) and restrains its K63-linked polyubiquitination, leading to attenuation of IKKi-mediated activation of NF-κB and repression of the phosphorylation and nuclear translocation of p65. Additionally, gp110 interacts with an important regulator of the Wnt signaling pathway, β-catenin, and induces its K48-linked polyubiquitination degradation via the proteasome system, resulting in the suppression of β-catenin-mediated IFN-β production. Taken together, these results suggest that gp110 is a negative regulator of antiviral immunity, revealing a novel mechanism of EBV immune evasion during lytic infection. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous pathogen that infects almost all human beings, and the persistence of EBV in the host is largely due to immune escape mediated by its encoded products. Thus, elucidation of EBV's immune escape mechanisms will provide a new direction for the design of novel antiviral strategies and vaccine development. Here, we report that EBV-encoded gp110 serves as a novel viral immune evasion factor, which inhibits RIG-I-like receptor pathway-mediated interferon-β (IFN-β) production. Furthermore, we found that gp110 targeted two key proteins, inhibitor of NF-κB kinase (IKKi) and β-catenin, which mediate antiviral activity and the production of IFN-β. gp110 inhibited K63-linked polyubiquitination of IKKi and induced β-catenin degradation via the proteasome, resulting in decreased IFN-β production. In summary, our data provide new insights into the EBV-mediated immune evasion surveillance strategy.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

SRSF3 (SRp20) is the smallest member of the serine/arginine (SR)-rich protein family. We found the annotated human SRSF3 and mouse Srsf3 RefSeq sequences are much larger than the detected SRSF3/Srsf3 RNA size by Northern blot. Mapping of RNA-seq reads from various human and mouse cell lines to the annotated SRSF3/Srsf3 gene illustrated only a partial coverage of its terminal exon 7. By 5' RACE and 3' RACE, we determined that SRSF3 gene spanning over 8422 bases and Srsf3 gene spanning over 9423 bases. SRSF3/Srsf3 gene has seven exons with exon 7 bearing two alternative polyadenylation signals (PAS). Through alternative PAS selection and exon 4 exclusion/inclusion by alternative RNA splicing, SRSF3/Srsf3 gene expresses four RNA isoforms. The major SRSF3 mRNA isoform with exon 4 exclusion by using a favorable distal PAS to encode a full-length protein is 1411 nt long (not annotated 4228 nt) and the same major mouse Srsf3 mRNA isoform is only 1295 nt (not annotated 2585 nt). The difference from the redefined RNA size of SRSF3/Srsf3 to the corresponding RefSeq sequence is at the 3' UTR region. Collectively, the redefined SRSF3/Srsf3 gene structure and expression will allow better understanding of SRSF3 functions and its regulations in health and diseases.