HIV and prior exposure to antiretroviral therapy alter tumour composition and tumour: T-cell associations in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma worldwide, accounting for up to 40% of new non-Hodgkin Lymphoma (NHL) globally. People living with HIV are up to 17 times more likely to develop NHL, and as such, DLBCL is the leading cause of cancer death in this high-risk population. While histologically indistinguishable, HIV-associated (HIV+) and HIV-negative (HIV-) DLBCL are molecularly distinct, and biological differences may have implications for the development of future therapeutic interventions. Further, the impact of immunologic differences in people with HIV, including preceding ART, remains largely unknown. Here, we investigate the impact of HIV infection and ART exposure on the clinical features of DLBCL and T-cell immune response by performing imaging mass cytometry on our unique patient cohort in Malawi. In this cohort, HIV infection is positively prognostic, and HIV+/ART-naïve patients have the best outcomes. No established biomarkers other than Ki67 are associated with HIV or ART status, and the only tumour-intrinsic biomarkers that remain prognostic are MYC and MYC/BCL2 protein co-expression. Finally, TCR clonality is associated with distinct tumour-T cell interactions by HIV/ART status, indicating differential anti-tumour immune responses. We demonstrate previously undescribed HIV and ART-related differences in the DLBCL tumour microenvironment.

The complete Kaposi sarcoma-associated herpesvirus genome induces early-onset, metastatic angiosarcoma in transgenic mice.

Kaposi sarcoma (KS) is the most common cancer in persons living with HIV. It is caused by KS-associated herpesvirus (KSHV). There exists no animal model for KS. Pronuclear injection of the 170,000-bp viral genome induces early-onset, aggressive angiosarcoma in transgenic mice. The tumors are histopathologically indistinguishable from human KS. As in human KS, all tumor cells express the viral latency-associated nuclear antigen (LANA). The tumors transcribe most viral genes, whereas endothelial cells in other organs only transcribe the viral latent genes. The tumor cells are of endothelial lineage and exhibit the same molecular pattern of pathway activation as KS, namely phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR, interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF). The KSHV-induced tumors are more aggressive than Ha-ras-induced angiosarcomas. Overall survival is increased by prophylactic ganciclovir. Thus, whole-virus KSHV-transgenic mice represent an accurate model for KS and open the door for the genetic dissection of KS pathogenesis and evaluation of therapies, including vaccines.

Inhibition of NEK2 Promotes Chemosensitivity and Reduces KSHV-positive Primary Effusion Lymphoma Burden.

Non-Hodgkin lymphoma (NHL) is a common cancer in both men and women and represents a significant cancer burden worldwide. Primary effusion lymphoma (PEL) is a subtype of NHL infected with Kaposi sarcoma-associated herpesvirus (KSHV). PEL is an aggressive and lethal cancer with no current standard of care, owing largely to its propensity to develop resistance to current chemotherapeutic regimens. Here, we report a reliance of KSHV-positive PEL on the mitotic kinase, NEK2, for survival. Inhibition of NEK2 with the inhibitor, JH295, resulted in caspase 3-mediated apoptotic cell death of PEL. Furthermore, NEK2 inhibition significantly prolonged survival and reduced tumor burden in a PEL mouse model. We also demonstrate that the ABC transporter proteins, MDR1 and MRP, are most active in PEL and that inhibition of NEK2 in PEL reduced the expression and activity of these ABC transporter proteins, which are known to mediate drug resistance in cancer. Finally, we report that JH295 treatment sensitized lymphomas to other chemotherapeutic agents such as rapamycin, resulting in enhanced cancer cell death. Overall, these data offer important insight into the mechanisms underlying PEL survival and drug resistance, and suggest that NEK2 is a viable therapeutic target for PEL. SIGNIFICANCE: The mitotic kinase, NEK2, is important for the survival of KSHV-positive PEL. NEK2 inhibition resulted in PEL apoptosis and reduced tumor burden in a mouse model. NEK2 inhibition also reduced drug resistance.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

AGC kinase inhibitors regulate STING signaling through SGK-dependent and SGK-independent mechanisms.

Type 1 IFN expression is critical in the innate immune response, but aberrant expression is associated with autoimmunity and cancer. Here, we identify N-[4-(1H46 pyrazolo[3,4-b] pyrazin-6-yl)-phenyl]-sulfonamide (Sanofi-14h), a compound with preference for inhibition of the AGC family kinase SGK3, as an inhibitor of Ifnb1 gene expression in response to STING stimulation of macrophages. Sanofi-14h abrogated SGK activity and also impaired activation of the critical TBK1/IRF3 pathway downstream of STING activation, blocking interaction of STING with TBK1. Deletion of SGK1/3 in a macrophage cell line did not block TBK1/IRF3 activation but decreased expression of transcription factors, such as IRF7 and STAT1, required for the innate immune response. Other AGC kinase inhibitors blocked TBK1 and IRF3 activation suggesting common action on a critical regulatory node in the STING pathway. These studies reveal both SGK-dependent and SGK-independent mechanisms in the innate immune response and indicate an approach to block aberrant Ifnb1 expression.

Kaposi's sarcoma-associated herpesvirus viral protein kinase augments cell survival.

Oncogenic viruses have developed various strategies to antagonize cell death and maintain lifelong persistence in their host, a relationship that may contribute to cancer development. Understanding how viruses inhibit cell death is essential for understanding viral oncogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three different cancers in the human population, including Kaposi's sarcoma (KS), the most common cancer in HIV patients. Previous studies have indicated that the KSHV-encoded viral protein kinase (vPK) impacts many processes dysregulated in tumorigenesis. Here, we report that vPK protects cells from apoptosis mediated by Caspase-3. Human umbilical vein endothelial cells (HUVECs) expressing vPK (HUVEC-vPK) have a survival advantage over control HUVEC under conditions of extrinsic- and intrinsic-mediated apoptosis. Abolishing the catalytic activity of vPK attenuated this survival advantage. We found that KSHV vPK-expressing HUVECs exhibited increased activation of cellular AKT kinase, a cell survival kinase, compared to control cells without vPK. In addition, we report that vPK directly binds the pleckstrin homology (PH) domain of AKT1 but not AKT2 or AKT3. Treatment of HUVEC-vPK cells with a pan-AKT inhibitor Miransertib (ARQ 092) reduced the overall phosphorylation of AKT, resulting in the cleavage of Caspase-3 and the induction of apoptosis. Furthermore, vPK expression activated VEGF/VEGFR2 in HUVECs and promoted angiogenesis through the AKT pathway. vPK expression also inhibited the cytotoxicity of cisplatin in vitro and in vivo. Collectively, our findings demonstrate that vPK's ability to augment cell survival and promote angiogenesis is critically dependent on AKT signaling, which is relevant for future therapies for treating KSHV-associated cancers.

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.

A prospective cohort study identifies two types of HIV+ Kaposi Sarcoma lesions: proliferative and inflammatory.

Kaposi sarcoma (KS) is the most common cancer in people living with HIV (PLWH) in many countries where KS-associated herpesvirus is endemic. Treatment has changed little in 20 years, but the disease presentation has. This prospective cohort study enrolled 122 human immunodeficiency virus (HIV) positive KS patients between 2017 and 2019 in Malawi. Participants were treated with bleomycin, vincristine and combination antiretroviral therapy, the local standard of care. One-year overall survival was 61%, and progression-free survival was 58%. The 48-week complete response rate was 35%. RNAseq (n = 78) differentiated two types of KS lesions, those with marked endothelial characteristics and those enriched in inflammatory transcripts. This suggests that different KS lesions are in different disease states consistent with the known heterogeneous clinical response to treatment. In contrast to earlier cohorts, the plasma HIV viral load of KS patients in our study was highly variable. A total of 25% of participants had no detectable HIV; all had detectable KSHV viral load. Our study affirms that many KS cases today develop in PLWH with well-controlled HIV infection and that different KS lesions have differing molecular compositions. Further studies are needed to develop predictive biomarkers for this disease.

The Role of RNA Sensors in Regulating Innate Immunity to Gammaherpesviral Infections.

Kaposi's sarcoma-associated herpesvirus (KSHV) and the Epstein-Barr virus (EBV) are double-stranded DNA oncogenic gammaherpesviruses. These two viruses are associated with multiple human malignancies, including both B and T cell lymphomas, as well as epithelial- and endothelial-derived cancers. KSHV and EBV establish a life-long latent infection in the human host with intermittent periods of lytic replication. Infection with these viruses induce the expression of both viral and host RNA transcripts and activates several RNA sensors including RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), protein kinase R (PKR) and adenosine deaminases acting on RNA (ADAR1). Activation of these RNA sensors induces the innate immune response to antagonize the virus. To counteract this, KSHV and EBV utilize both viral and cellular proteins to block the innate immune pathways and facilitate their own infection. In this review, we summarize how gammaherpesviral infections activate RNA sensors and induce their downstream signaling cascade, as well as how these viruses evade the antiviral signaling pathways to successfully establish latent infection and undergo lytic reactivation.

Development of VHL-recruiting STING PROTACs that suppress innate immunity.

STING acts as a cytosolic nucleotide sensor to trigger host defense upon viral or bacterial infection. While STING hyperactivation can exert anti-tumor effects by increasing T cell filtrates, in other contexts hyperactivation of STING can contribute to autoimmune and neuroinflammatory diseases. Several STING targeting agonists and a smaller subset of antagonists have been developed, yet STING targeted degraders, or PROTACs, remain largely underexplored. Here, we report a series of STING-agonist derived PROTACs that promote STING degradation in renal cell carcinoma (RCC) cells. We show that our STING PROTACs activate STING and target activated/phospho-STING for degradation. Locking STING on the endoplasmic reticulum via site-directed mutagenesis disables STING translocation to the proteasome and resultingly blocks STING degradation. We also demonstrate that PROTAC treatment blocks downstream innate immune signaling events and attenuates the anti-viral response. Interestingly, we find that VHL acts as a bona fide E3 ligase for STING in RCC; thus, VHL-recruiting STING PROTACs further promote VHL-dependent STING degradation. Our study reveals the design and biological assessment of VHL-recruiting agonist-derived STING PROTACs, as well as demonstrates an example of hijacking a physiological E3 ligase to enhance target protein degradation via distinct mechanisms.

Today's Kaposi sarcoma is not the same as it was 40 years ago, or is it?

This review will provide an overview of the notion that Kaposi sarcoma (KS) is a disease that manifests under diverse and divergent circumstances. We begin with a historical introduction of KS and KS-associated herpesvirus (KSHV), highlight the diversity of clinical presentations of KS, summarize what we know about the cell of origin for this tumor, explore KSHV viral load as a potential biomarker for acute KSHV infections and KS-associated complications, and discuss immune modulators that impact KSHV infection, KSHV persistence, and KS disease.

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

Molecular interactions between viral DNA and viral-encoded protein are a prerequisite for successful herpesvirus replication and production of new infectious virions. Here, we examined how the essential Kaposi's sarcoma-associated herpesvirus (KSHV) protein, RTA, binds to viral DNA using transmission electron microscopy (TEM). Previous studies using gel-based approaches to characterize RTA binding are important for studying the predominant form(s) of RTA within a population and identifying the DNA sequences that RTA binds with high affinity. However, using TEM we were able to examine individual protein-DNA complexes and capture the various oligomeric states of RTA when bound to DNA. Hundreds of images of individual DNA and protein molecules were collected and then quantified to map the DNA binding positions of RTA bound to the two KSHV lytic origins of replication encoded within the KSHV genome. The relative size of RTA or RTA bound to DNA were then compared to protein standards to determine whether RTA complexed with DNA was monomeric, dimeric, or formed larger oligomeric structures. We successfully analyzed a highly heterogenous dataset and identified new binding sites for RTA. This provides direct evidence that RTA forms dimers and high order multimers when bound to KSHV origin of replication DNA sequences. This work expands our understanding of RTA binding, and demonstrates the importance of employing methodologies that can characterize highly heterogenic populations of proteins. 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.

KSHV Viral Protein Kinase Interacts with USP9X to Modulate the Viral Lifecycle.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X.

Epstein-Barr Virus-Positive Lymphomas Exploit Ectonucleotidase Activity To Limit Immune Responses and Prevent Cell Death.

Epstein-Barr virus (EBV) is a cancer-associated virus that infects more than 90% of adults. Unfortunately, many EBV-driven malignancies, including numerous B cell lymphomas, are highly aggressive and lack acceptable therapeutic outcomes. The concentrations of extracellular purines, namely, ATP and adenosine, are highly dysregulated in the tumor microenvironment and significantly impact the degree of immune responses to the tumor. Additionally, many tumor cells adapt to this dysregulation by overexpressing one or more ectonucleotidases, enzymes that degrade extracellular nucleotides to nucleosides. The degradation of immunostimulatory extracellular ATP to immunosuppressive adenosine through ectonucleotidase activity is one example of tumor cell exploitation of the purinergic signaling pathway. As such, preclinical studies targeting the purinergic signaling pathway have found it to be a promising immunotherapeutic target for the treatment of solid tumors; however, the extent to which purinergic signaling impacts the development and survival of EBV+ B cell lymphoma remains unstudied. Here, we demonstrate robust ectonucleotidase expression on multiple types of EBV-positive B cell non-Hodgkin lymphoma (NHL). Furthermore, the presence of high concentrations of extracellular ATP resulted in the expression of lytic viral proteins and exhibited cytotoxicity toward EBV+ B cell lines, particularly when CD39 was inhibited. Inhibition of CD39 also significantly prolonged survival in an aggressive cord blood humanized mouse model of EBV-driven lymphomagenesis and was correlated with an enhanced inflammatory immune response and reduced tumor burden. Taken together, these data suggest that EBV+ B cell lymphomas exploit ectonucleotidase activity to circumvent ATP-mediated inflammation and cell death. IMPORTANCE EBV is a ubiquitous pathogen responsible for significant global lymphoma burden, including Hodgkin lymphoma, numerous non-Hodgkin B, T, and NK cell lymphomas, and lymphoproliferative disorders. EBV is also associated with epithelial cancers and autoimmune diseases, such as multiple sclerosis. Many of these diseases are highly aggressive and exhibit poor outcomes. As such, new treatments for EBV-driven cancers have the potential to benefit a large number of patients. We use in vitro and in vivo models to demonstrate the therapeutic potential of targeting the purinergic signaling pathway in the context of EBV-driven B cell lymphoma. These findings lend credence to the manipulation of purinergic signaling as a viable therapeutic approach to EBV+ malignancies and support the feasibility of immunotherapeutic treatments for viral lymphoma.

Intra-Host Evolution Provides for the Continuous Emergence of SARS-CoV-2 Variants.

Variants of concern (VOC) in SARS-CoV-2 refer to viruses whose viral genomes differ from the ancestor virus by ≥3 single-nucleotide variants (SNVs) and that show the potential for higher transmissibility and/or worse clinical progression. VOC have the potential to disrupt ongoing public health measures and vaccine efforts. Still, too little is known regarding how frequently new viral variants emerge and under what circumstances. We report a study to determine the degree of SARS-CoV-2 sequence evolution in 94 patients and to estimate the frequency at which highly diverse variants emerge. Two cases accumulated ≥9 SNVs over a 2-week period and one case accumulated 23 SNVs over 3 weeks, including three nonsynonymous mutations in the spike protein (D138H, E554D, D614G). The remainder of the infected patients did not show signs of intra-host evolution. We estimate that in as much as 2% of hospitalized COVID-19 cases, variants with multiple mutations in the spike glycoprotein emerge in as little as 1 month of persistent intra-host virus replication. This suggests the continued local emergence of variants with multiple nonsynonymous SNVs, even in patients without overt immune deficiency. Surveillance by sequencing for (i) viremic COVID-19 patients, (ii) patients suspected of reinfection, and (iii) patients with diminished immune function may offer broad public health benefits. IMPORTANCE New SARS-CoV-2 variants can potentially disrupt ongoing public health measures and vaccine efforts. Still, little is known regarding how frequently new viral variants emerge and under what circumstances. Based on this study, we estimate that in hospitalized COVID-19 cases, variants with multiple mutations may emerge locally in as little as 1 month, even in patients without overt immune deficiency. Surveillance by sequencing for continuously shedding patients, patients suspected of reinfection, and patients with diminished immune function may offer broad public health benefits.

Barrier-to-autointegration factor 1 promotes gammaherpesvirus reactivation from latency.

Gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are DNA viruses that are globally associated with human cancers and establish lifelong latency in the human population. Detection of gammaherpesviral infection by the cGAS-STING innate immune DNA-sensing pathway is critical for suppressing viral reactivation from latency, a process that promotes viral pathogenesis and transmission. We report that barrier-to-autointegration factor 1 (BAF)-mediated suppression of the cGAS-STING signaling pathway is necessary for reactivation of KSHV and EBV. We demonstrate a role for BAF in destabilizing cGAS expression and show that inhibiting BAF expression in latently infected, reactivating, or uninfected cells leads to increased type I interferon-mediated antiviral responses and decreased viral replication. Furthermore, BAF overexpression resulted in decreased cGAS expression at the protein level. These results establish BAF as a key regulator of the lifecycle of gammaherpesviruses and a potential target for treating viral infections and malignancies.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.