A virus-induced circular RNA maintains latent infection of Kaposi's sarcoma herpesvirus.

Non-coding RNAs (ncRNAs) play important roles in host-pathogen interactions; oncogenic viruses like Kaposi's sarcoma herpesvirus (KSHV) employ ncRNAs to establish a latent reservoir and persist for the life of the host. We previously reported that KSHV infection alters a novel class of RNA, circular RNAs (circRNAs). CircRNAs are alternative splicing isoforms and regulate gene expression, but their importance in infection is largely unknown. Here, we showed that a human circRNA, hsa_circ_0001400, is induced by various pathogenic viruses, namely KSHV, Epstein-Barr virus, and human cytomegalovirus. The induction of circRNAs including circ_0001400 by KSHV is co-transcriptionally regulated, likely at splicing. Consistently, screening for circ_0001400-interacting proteins identified a splicing factor, PNISR. Functional studies using infected primary endothelial cells revealed that circ_0001400 inhibits KSHV lytic transcription and virus production. Simultaneously, the circRNA promoted cell cycle, inhibited apoptosis, and induced immune genes. RNA-pull down assays identified transcripts interacting with circ_0001400, including TTI1, which is a component of the pro-growth mTOR complexes. We thus identified a circRNA that is pro-growth and anti-lytic replication. These results support a model in which KSHV induces circ_0001400 expression to maintain latency. Since circ_0001400 is induced by multiple viruses, this novel viral strategy may be widely employed by other viruses.

The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

KSHV genome harbors both constitutive and lytically induced enhancers.

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.

Electron microscopy mapping of the DNA-binding sites of monomeric, dimeric, and multimeric KSHV RTA protein.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus associated with several human cancers, typically in patients with compromised immune systems. Herpesviruses establish lifelong infections in hosts in part due to the two phases of infection: the dormant and active phases. Effective antiviral treatments to prevent the production of new viruses are needed to treat KSHV. A detailed microscopy-based investigation of the molecular interactions between viral protein and viral DNA revealed how protein-protein interactions play a role in DNA-binding specificity. This analysis will lead to a more in-depth understanding of KSHV DNA replication and serve as the basis for anti-viral therapies that disrupt and prevent the protein-DNA interactions, thereby decreasing spread to new hosts.

Molecular epidemiology of human herpesvirus 8 in patients with HHV-8-related diseases in Ireland.

Human Herpesvirus 8 (HHV-8) has been classified by sequence analysis of open reading frame (ORF) K1, ORF K15, and variable sequence loci within the central constant region. The purpose of this study was to examine the molecular epidemiology of HHV-8 in an Irish population. This retrospective study included 30 patients who had HHV-8 DNA detected in plasma. Nested end-point PCR was used to characterise four regions of the HHV-8 genome, K1, T0.7 (K12), ORF 75, and K15. Sequencing data were obtained for 23 specimens from 19 patients. Phylogenetic analysis of ORF K1 demonstrated that subtypes A, B, C and F were present in 37%, 11%, 47% and 5%, respectively. For T0.7 and ORF 75, sequencing data were obtained for 12 patients. For T0.7, subtypes A/C, J, B, R and Q were present in 58%, 17%, 8%, 8%, and 8%, respectively. For ORF 75, subtypes A, B, C and D were present in 58%, 8%, 25%, and 8%, respectively. K15 sequences were determined for 13 patients. 69% had the P allele and 31% had the M allele. The data generated by this study demonstrate that a broad variety of HHV-8 subtypes are represented in patients exhibiting HHV-8-related disease in Ireland, a low prevalence country. The predominance of C and A K1 subtypes was as expected for a Western European population. The 31% prevalence for K15 subtype M was higher than expected for a Western European population. This may represent the changing and evolving epidemiology in Ireland due to altered migration patterns.

Combined Flow-Fluorescence in situ hybridization to HHV-8 and EBV reveals the viral heterogeneity of primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare B-cell non-Hodgkin lymphoma associated with Kaposi Sarcoma-associated herpesvirus (KSHV/HHV8) infection. Lymphoma cells are coinfected with Epstein-Barr virus (EBV) in 60-80% of cases. Tools allowing a reliable PEL diagnosis are lacking. This study reports PEL diagnosis in 4 patients using a Flow-Fluorescence in situ hybridization (FlowFISH) technique that allowed detection of differentially expressed EBV and HHV8 transcripts within the same sample, revealing viral heterogeneity of the disease. Moreover, infected cells exhibited variable expressions of CD19, CD38, CD40, and CD138. Therefore, FlowFISH is a promising tool to diagnose and characterize complex viral lymphoproliferations.

A role of BPTF in viral oncogenicity delineated through studies of heritable Kaposi sarcoma.

Kaposi sarcoma (KS), caused by Herpesvirus-8 (HHV-8; KSHV), shows sporadic, endemic, and epidemic forms. While familial clustering of KS was previously recorded, the molecular basis of hereditary predilection to KS remains largely unknown. We demonstrate through genetic studies that a dominantly inherited missense mutation in BPTF segregates with a phenotype of classical KS in multiple immunocompetent individuals in two families. Using an rKSHV.219-infected CRISPR/cas9-model, we show that BPTFI2012T mutant cells exhibit higher latent-to-lytic ratio, decreased virion production, increased LANA staining, and latent phenotype in viral transcriptomics. RNA-sequencing demonstrated that KSHV infection dysregulated oncogenic-like response and P53 pathways, MAPK cascade, and blood vessel development pathways, consistent with KS. BPTFI2012T also enriched pathways of viral genome regulation and replication, immune response, and chemotaxis, including downregulation of IFI16, SHFL HLAs, TGFB1, and HSPA5, all previously associated with KSHV infection and tumorigenesis. Many of the differentially expressed genes are regulated by Rel-NF-κB, which regulates immune processes, cell survival, and proliferation and is pivotal to oncogenesis. We thus demonstrate BPTF mutation-mediated monogenic hereditary predilection of KSHV virus-induced oncogenesis, and suggest BPTF as a drug target.

Angioimmunoblastic T-cell lymphoma and Kaposi sarcoma: A fortuitous collision?

AIMS: Follicular helper T-cell (TFH) lymphoma of the angioimmunoblastic-type (AITL), one of the most prevalent T-cell lymphomas, typically encompasses proliferation of high endothelial venules and Epstein-Barr virus-positive immunoblasts, but neither infection with HHV8 nor association with Kaposi's sarcoma (KS) have been described. The aims of this study are to characterise the association between AITL and HHV8 infection or KS. METHODS AND RESULTS: Three male patients aged 49-76 years, HIV-negative, with concurrent nodal involvement by AITL and KS, were identified from our files and carefully studied. Two patients originated from countries where endemic KS occurs, including one with cutaneous KS. The lymphomas featured abundant vessels, expanded follicular dendritic cells and neoplastic TFH cells [PD1+ (three of three), ICOS+ (three of three), CXCL13+ (three of three), CD10+ (two of three), BCL6 (two of three)] but lacked EBV+ immunoblasts. The foci of KS consisted of subcapsular proliferations of ERG+, CD31+ and/or CD34+ , HHV8+ spindle cells. High-throughput sequencing showed AITL-associated mutations in TET2 (three of three), RHOA (G17V) (three of three) and IDH2 (R172) (two of three), which were absent in the microdissected KS component in two cases. Relapses in two patients consisted of AITL, without evidence of KS. No evidence of HHV8 infection was found in a control group of 23 AITL cases. CONCLUSION: Concurrent nodal involvement by AITL and KS is rare and identification of both neoplastic components may pose diagnostic challenges. The question of whether the association between AITL and KS may be fortuitous or could reflect the underlying immune dysfunction in AITL remains open.

A variant of KSHV-associated inflammatory cytokine syndrome in elderly men of Mediterranean descent.

The spectrum of HHV-8-associated disorders includes Kaposi's sarcoma, primary effusion lymphoma, multicentric Castleman's disease, and the recently described KSHV inflammatory cytokine syndrome (KICS), a life-threatening disorder complicating HIV infection. There have been no reports in the literature concerning non-immunosuppressed individuals affected with KICS. We report here a KICS-like illness occurring in two elderly Greek men without HIV infection or other recognizable cause of immunosuppression.

The Cytokine Storm of Multicentric Castleman Disease.

As described throughout this book, different triggers can elicit a variety of different cytokine storm disorders that share overlapping clinical features (Fig. 31.1). Even within a particular cytokine storm disorder, multiple different triggers can elicit the syndrome. Like HLH, multicentric Castleman disease (MCD) serves as a great example of this as it can be caused by a viral infection, neoplastic cell population, or an unknown cause. Furthermore, the idiopathic subtype of MCD (iMCD) provides one of the first examples of a cytokine storm disorder that could be abrogated with targeted neutralization of a single cytokine when inhibition with the anti-interleukin-6 (IL-6) receptor monoclonal antibody tocilizumab was shown to effectively treat iMCD in the 1990s. Of course, this "iMCD treatment," tocilizumab, has been used in a variety of cytokine storm settings over the last 30+ years.

Prognostic factors in Kaposi sarcoma, single centre experience.

BACKGROUND: Kaposi sarcoma (KS) is a multicentric vascular and lymphatic neoplasm caused by human herpesvirus 8 (HHV-8). It generally concerns the elderly and immunosuppressed population. Four major clinical types of KS have been described. The most common subtype is Classical KS (CKS). OBJECTIVES: Our retrospective study aimed to better define prognostic subgroups among patients with CKS, which is the most common in our country. METHOD: Between 2014 and 2020, 43 patients with CKS were treated with local excision, radiotherapy and chemotherapy. Reviewed information included demographics, clinical features, laboratory findings, treatment responses and overall survival. RESULTS: During the follow-up, eight patients (18.6%) died of CKS. The complete response rate was 46.5%, partial response and stable disease 51.2%, and progressive disease 2.3% of all patients. Gender, haemoglobin level at diagnosis, and disseminated involvement were prognostic factors affecting survival in all patients. CONCLUSION: We confirmed that male gender, low haemoglobin levels, and disseminated involvement are associated with poor prognosis in CKS patients. It is the only Turkish study in which prognostic analysis was performed for this rare cancer.

Molecular Features of HHV8 Monoclonal Microlymphoma Associated with Kaposi Sarcoma and Multicentric Castleman Disease in an HIV-Negative Patient.

Human herpesvirus 8 (HHV8)-associated diseases include Kaposi sarcoma (KS), multicentric Castleman disease (MCD), germinotropic lymphoproliferative disorder (GLPD), Kaposi sarcoma inflammatory cytokine syndrome (KICS), HHV8-positive diffuse large B-cell lymphoma (HHV8+ DLBCL), primary effusion lymphoma (PEL), and extra-cavitary PEL (ECPEL). We report the case of a human immunodeficiency virus (HIV)-negative male treated for cutaneous KS, who developed generalized lymphadenopathy, hepatosplenomegaly, pleural and abdominal effusions, renal insufficiency, and pancytopenia. The excised lymph node showed features of concomitant involvement by micro-KS and MCD, with aggregates of HHV8+, Epstein Barr virus (EBV)-negative, IgM+, and lambda+ plasmablasts reminiscent of microlymphoma. Molecular investigations revealed a somatically hypermutated (SHM) monoclonal rearrangement of the immunoglobulin heavy chain (IGH), accounting for 4% of the B-cell population of the lymph node. Mutational analyses identified a pathogenic variant of KMT2D and variants of unknown significance in KMT2D, FOXO1, ARID1A, and KMT2A. The patient died shortly after surgery. The histological features (HHV8+, EBV-, IgM+, Lambda+, MCD+), integrated with the molecular findings (monoclonal IGH, SHM+, KMT2D mutated), supported the diagnosis of a monoclonal HHV8+ microlymphoma, with features intermediate between an incipient HHV8+ DLBCL and an EBV-negative ECPEL highlighting the challenges in the accurate classification of HHV8-driven lymphoid proliferations.

Monomorphic T-cell post-transplant lymphoproliferative disorder with features of HHV8-negative primary effusion lymphoma: an autopsy case and review of the literature.

A 67-year-old man underwent renal transplantation in his twenties. He developed refractory pleural effusion, with many large lymphocytes with severe atypia and mitosis in the effusion, indicating malignant lymphoma. He finally died of respiratory failure. An autopsy revealed atypical lymphocytes positive for CD3, CD4, and CD30 and negative for CD8, CD20, PAX5, human herpesvirus (HHV) 8, and Epstein-Barr virus-encoded small RNAs by immunohistochemistry and in situ hybridization. Atypical lymphocytes also had T-cell receptor gene rearrangements Jß2, Jγ2, and Jδ1 and chromosomal aberrations der(8)t(1;8)(q21;p21), add(13)(q12), add(14)(q32), and add(16)(q12-13). A few atypical lymphocytes were present at other sites. We finally diagnosed this case as monomorphic T-cell post-transplant lymphoproliferative disorder with features of HHV8-negative primary effusion lymphoma. A literature review only identified six cases (four HHV8-negative, two HHV8-positive) of effusion lymphoma of T-cell type, including the present case. Interestingly, about half of HHV8-negative and HHV8-positive cases had a history of renal transplantation in their twenties. All cases showed tumor CD30 expression, whereas CD4 and CD8 expressions were inconsistent. These findings indicated that this lymphoma may be associated with post-transplant lymphoproliferative disorder by renal transplantation at a young age, although further cases need to be analyzed.

Prevalence, Risk factors, and Shedding of Human Herpes Virus-8 among Men Having Sex With Men Enrolled in a Pre-exposure Prophylaxis Study.

BACKGROUND: Seroprevalence and risk factors for Human Herpesvirus-8 (HHV-8) infection among HIV-negative men who have sex with men (MSM) on pre-exposure prophylaxis (PrEP) have not been well characterized. Our objectives were to assess the prevalence and incidence of HHV-8 infection in MSM enrolled on PrEP and assess viral shedding in seropositive participants. METHODS: The ANRS IPERGAY study enrolled 429 participants in France and Canada to evaluate oral PrEP for HIV-1 prevention. Stored sera samples at day 0 (D0) and last visit were tested for the detection of HHV-8 antibodies using an indirect immunofluorescence assay. Baseline characteristics were analyzed to identify risk factors associated with HHV-8 seropositivity. Among seropositive participants, HHV-8 DNA was quantified on available oral and anal swabs, and ORF-K1 typing performed on HHV-8 positive samples. RESULTS: One hundred participants were seropositive at D0 (prevalence of 24%, 95% Confidence Interval (95%CI): 20·0-28·4) and 18/329 seroconverted during the study (incidence rate of 2·66 per 100 person-years, 95%CI: 1·57-4·20). Risk factors independently associated with baseline HHV-8 seropositivity included older age, high number of sexual partners, chemsex use and HSV-2 seropositivity. Among HHV-8 seropositive participants with available swab(s) for virological analysis, 37/115 (32%) displayed HHV-8 oral shedding, and 5/113 (4.4%) anal shedding at least once. Four patients had positive viral load before seroconversion. CONCLUSION: Prevalence and incidence of HHV-8 infection were high in HIV-negative PrEP users. Among seropositive participants, HHV-8 DNA is mainly detected in saliva, which may play a major role in viral transmission in this population.

Evaluation of human herpesvirus-8 viremia and antibody positivity in patients with HIV infection with human herpesvirus-8-related diseases.

Human herpesvirus-8 (HHV-8) viremia is associated with refractory conditions in patients infected with HIV-1. Therefore, we evaluated the factors related to plasma HHV-8-DNA. Participants included patients infected with HIV-1 who visited our hospital. Plasma HHV-8-DNA levels were measured using real-time polymerase chain reaction, and anti-HHV-8 antibodies were assessed through enzyme immunoassays using multiple antigens (K8.1, ORF59, ORF65, and LANA). Factors related to plasma HHV-8-DNA were examined using Fisher's exact test or Mann-Whitney U test. The study involved 36 patients infected with HIV-1, of whom 19 were histologically diagnosed with Kaposi's sarcoma (KS), two had multicentric Castleman's disease (MCD), and 15 did not exhibit HHV-8-related disease. Before the introduction of antiretroviral therapy (ART), plasma HHV-8-DNA was detected in 44% (7/16) of patients with KS and in 9% (1/11) of patients without HHV-8-related disease. Among patients with KS, elevated plasma HHV-8-DNA levels (≥0.05 copies/µL) correlated with the presence of CDC category C diseases other than KS (p = 0.0337), anti-HHV-8 antibody negativity (p = 0.0337), anemia (p = 0.0474), and thrombocytopenia (p = 0.0146). Following ART initiation, the percentage of patients positive for plasma HHV-8-DNA decreased from 44% (7/16) to 6% (1/17), and the percentage of patients positive for anti-HHV-8 antibodies increased from 44% (7/16) to 88% (15/17). Finally, plasma HHV-8-DNA positivity and anti-HHV-8 antibody negativity were observed in two patients with MCD. Our findings suggest that insufficient production of anti-HHV-8 antibodies was associated with HHV-8 viremia, and that anti-HHV-8 antibody production was recovered with ART; thus, indicating the possibility of involvement of humoral immunity in suppressing HHV-8 viremia.

Gastric ulcer and duodenitis associated with coinfection of human herpesvirus-8 and cytomegalovirus in a renal transplant recipient: a case report.

BACKGROUND: The coinfection between cytomegalovirus (CMV) and either human herpesvirus-6 (HHV-6) or HHV-7 in renal transplant recipients is well known; however, there have been few reports of coinfection of CMV associated with HHV-8. This paper presents a first case of acute gastric ulcer and duodenitis associated with CMV and HHV-8 coinfection after renal transplantation. CASE PRESENTATION: A 33-year-old male with a history of kidney transplantation was admitted to hospital because of postural epigastric pain. The recipient was CMV seropositive prior to transplantation and received trimethoprim-sulfamethoxazole without universal prophylaxis. Approximately 5 months after renal transplant, the recipient complained postural epigastric pain. An endoscopy revealed diffuse ulcerative lesions in the lower body and in the antrum of the stomach, as well as several erythematous mucosal lesions in the duodenum. Histopathologic examination identified CMV inclusions consistent with invasive CMV disease and immunohistochemical staining showed positive results for HHV-8 and CMV. No tumorous diseases such as Kaposi's sarcoma were detected. After 3 weeks of intravenous ganciclovir treatment, we observed that serum CMV PCR remained within the normal range and clinical symptoms improved. A follow-up endoscopy performed 3 weeks later showed that the severity of the above mentioned lesions had improved. CONCLUSIONS: We report the first case of a renal transplant recipient diagnosed with acute gastric ulcer and duodenitis associated with coinfection of CMV and HHV-8. Ganciclovir appears to be effective in diseases associated with coinfection of CMV and HHV-8.

Changing therapeutic landscape in advanced Kaposi sarcoma: Current state and future directions.

Kaposi sarcoma is a malignant neoplasm arising from the endothelial cell lining of blood and lymphatic vessels. Herein, we discuss etiopathogenesis, clinical presentation, diagnostic criteria, updated guideline-based approach to its management and newer experimental approaches. Given its efficacy and side effect profile, pegylated doxorubicin is the currently preferred first-line therapy in advanced disease. Paclitaxel remains an alternative first-line option. At the time of relapse, patients can be retreated with the same agents as they often maintain their clinical efficacy. New therapeutic options are on the rise, with pomalidomide being approved in 2020 as a second-line therapy. Optimal control of retroviral infection in human immunodeficiency virus (HIV) positive is instrumental in preventing disease occurrence in most patients. Suppressing human herpes virus type 8 (HHV-8) infection might also play a role in controlling Kaposi sarcoma growth, yet clinical trials are lacking. Unraveling the molecular and genetic intricacies of Kaposi sarcoma's pathogenesis might allow for the emergence of novel and effective therapeutic strategies. Clinical trials are currently underway to establish potential roles for various targeted agents, immune checkpoint inhibitors (ICIs) and experimental agents in the treatment of advanced Kaposi sarcoma.

A CRISPR-Cas9 screen identifies mitochondrial translation as an essential process in latent KSHV infection of human endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The main proliferating component of KS tumors is a cell of endothelial origin termed the spindle cell. Spindle cells are predominantly latently infected with only a small percentage of cells undergoing viral replication. As there is no direct treatment for latent KSHV, identification of host vulnerabilities in latently infected endothelial cells could be exploited to inhibit KSHV-associated tumor cells. Using a pooled CRISPR-Cas9 lentivirus library, we identified host factors that are essential for the survival or proliferation of latently infected endothelial cells in culture, but not their uninfected counterparts. Among the many host genes identified, there was an enrichment in genes localizing to the mitochondria, including genes involved in mitochondrial translation. Antibiotics that inhibit bacterial and mitochondrial translation specifically inhibited the expansion of latently infected endothelial cells and led to increased cell death in patient-derived PEL cell lines. Direct inhibition of mitochondrial respiration or ablation of mitochondrial genomes leads to increased death in latently infected cells. KSHV latent infection decreases mitochondrial numbers, but there are increases in mitochondrial size, genome copy number, and transcript levels. We found that multiple gene products of the latent locus localize to the mitochondria. During latent infection, KSHV significantly alters mitochondrial biology, leading to enhanced sensitivity to inhibition of mitochondrial respiration, which provides a potential therapeutic avenue for KSHV-associated cancers.

T-Cells Subsets in Castleman Disease: Analysis of 28 Cases Including Unicentric, Multicentric and HHV8-Related Clinical Forms.

Castleman disease (CD) is a rare lymphoproliferative disorder that includes various clinico-pathological subtypes. According to clinical course, CD is divided into unicentric CD (UCD) and multicentric CD (MCD). MCD is further distinguished based on the etiological driver in herpes virus-8-related MCD (that can occur in the setting of HIV); in MCD associated with POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes); and idiopathic MCD (iMCD). The latter can also be divided in iMCD-TAFRO (thrombocytopenia, anasarca, fever, myelofibrosis, organomegaly) and iMCD not otherwise specified. To date, CD pathogenesis is still uncertain, but CD may represent the histological and clinical result of heterogeneous pathomechanisms. Transcriptome investigations in CD lymph nodes have documented the expression and up-regulation of different cytokines; furthermore, few recent studies have shown alterations of different T-cell subsets in CD patients, suggesting a possible role of the nodal microenvironment in CD development. On this basis, our study aimed to investigate the distribution of T-cell subsets in the clinico-pathological spectrum of CD. We evaluated the CD4/CD8 ratio and the number of T-regulatory (T-reg) FOXP3+ cells in 28 CD cases. In total, 32% of cases showed a decreased CD4/CD8 ratio due to increased CD8+ T-cells, including both UCD, iMCD, and HHV8+ MCD cases. The T-reg subset analysis revealed a statistically significant (p < 0.0001) lower mean number of FOXP3+ T-reg cells in CD cases when compared with non-specific reactive lymph nodes. We did not find statistically significant differences in T-reg numbers between the different CD subtypes. These findings may suggest that alterations in T-cell subpopulations that can lead to disruption of immune system control may contribute to the numerous changes in different cellular compartments that characterize CD.

The Impact of Co-Infections for Human Gammaherpesvirus Infection and Associated Pathologies.

The two oncogenic human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause significant disease burden, particularly in immunosuppressed individuals. Both viruses display latent and lytic phases of their life cycle with different outcomes for their associated pathologies. The high prevalence of infectious diseases in Sub-Saharan Africa (SSA), particularly HIV/AIDS, tuberculosis, malaria, and more recently, COVID-19, as well as their associated inflammatory responses, could potentially impact either virus' infectious course. However, acute or lytically active EBV and/or KSHV infections often present with symptoms mimicking these predominant diseases leading to misdiagnosis or underdiagnosis of oncogenic herpesvirus-associated pathologies. EBV and/or KSHV infections are generally acquired early in life and remain latent until lytic reactivation is triggered by various stimuli. This review summarizes known associations between infectious agents prevalent in SSA and underlying EBV and/or KSHV infection. While presenting an overview of both viruses' biphasic life cycles, this review aims to highlight the importance of co-infections in the correct identification of risk factors for and diagnoses of EBV- and/or KSHV-associated pathologies, particularly in SSA, where both oncogenic herpesviruses as well as other infectious agents are highly pervasive and can lead to substantial morbidity and mortality.