Biology subjects

Damania, B.

Publications and source records attributed to Damania, B..

4 recordsLinked to original sources

bioRxiv2026

Kaposi Sarcoma herpesvirus reactivation and replication are dispensable for cell-lineage specific tumorigenesis in whole genome transgenic mice

Herpesvirus reactivation and replication depend on an ordered cascade of gene expression. The viral immediate-early replication and transcription activator protein (Rta) is necessary and sufficient to initiate this process. For Kaposi sarcoma herpesvirus (KSHV), Rta-interacting and Rta-regulated proteins are thought to contribute to cellular transformation. Using a novel, whole-genome transgenic mouse model, we show that KSHV tumorigenesis is independent of Rta in vivo. Rta-deleted, KSHV bacmid transgenic (tgKSHV{Delta}Rta) mice developed latency-associated nuclear antigen (LANA)-positive vascular sarcomas with the same endothelial cell-lineage composition and transcriptional profile as human Kaposi sarcoma (KS). The viral miRNAs were expressed in the tumors, as were all canonical latency and the K1 and K15 oncogenes. Importantly, this system is the first to model both KS and lymphoma, reflecting the development of both malignancies observed in KSHV-infected individuals. These results separate virus replication from KS tumorigenesis, which can proceed independently so long as the virus genome is maintained in a susceptible cell. SignificanceKaposi sarcoma (KS) is the most common cancer in people living with HIV worldwide. Like all herpesviruses, KS-associated herpesvirus (KSHV) has a latent and lytic phase. Hitherto, viral lytic genes are thought to play a role in KS. The KSHV replication and transcription activator (Rta) is both necessary and sufficient for reactivation and lytic viral replication, but Rta was not required for vascular tumor development in a whole virus genome mouse model. This new tgKSHV{Delta}RTA mouse model separates virus replication from viral tumorigenesis and defines a minimal set of viral oncogenes. This model enables mechanistic studies in vivo, and treatment modalities to be evaluated within a complete immune and stromal microenvironment.

Cancer Biology↗

bioRxiv2023

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 Kaposis 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. ImportanceKaposis 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 biology↗

bioRxiv2022

AGC Kinase Inhibitors Regulate STING Signaling Through SGK-Dependent and SGK-Independent Mechanisms

The STING signaling pathway is essential for the innate immune response to DNA viruses and bacteria and is important in tumor immunity. STING binding to cGAMP or to synthetic agonists leads to the activation of the kinase TBK1 which phosphorylates the transcription factor IRF3 which promotes expression of type 1 interferons such as IFN{beta} to block viral activity. Aberrant type 1 IFN expression is associated with human diseases including autoimmunity, HIV, and cancer. Here we identify N-[4-(1H-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 IFN{beta} 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, notably blocking the ligand-induced interaction of STING with TBK1. Deletion of SGK1 and SGK3 in macrophages suppressed activation of IFN{beta} transcription but did not block TBK1/IRF3 activation downstream of STING. Gene and protein expression analysis revealed that deletion of SGK1/3 in a macrophage cell line decreases basal expression of critical transcription factors required for the innate immune response, such as IRF7 and STAT1. Additional studies reveal that other AGC kinase inhibitors block TBK1 and IRF3 activation suggesting common action on a critical regulatory node in the STING pathway. Thus, studies with Sanofi-14h have revealed both SGK-dependent and SGK-independent effects in the STING pathway and suggest a mechanism to alter type 1 IFN transcription through small molecule therapy.

immunology↗

bioRxiv2020

High-density amplicon sequencing identifies community spread and ongoing evolution of SARS-CoV-2 in the Southern United States

SARS-CoV-2 is constantly evolving. Prior studies have focused on high case-density locations, such as the Northern and Western metropolitan areas in the U.S. This study demonstrates continued SARS-CoV-2 evolution in a suburban Southern U.S. region by high-density amplicon sequencing of symptomatic cases. 57% of strains carried the spike D614G variant. The presence of D614G was associated with a higher genome copy number and its prevalence expanded with time. Four strains carried a deletion in a predicted stem loop of the 3 untranslated region. The data are consistent with community spread within the local population and the larger continental U.S. No strain had mutations in the target sites used in common diagnostic assays. The data instill confidence in the sensitivity of current tests and validate "testing by sequencing" as a new option to uncover cases, particularly those not conforming to the standard clinical presentation of COVID-19. This study contributes to the understanding of COVID-19 by providing an extensive set of genomes from a non-urban setting and further informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the U.S.

microbiology↗