Kaposi’s sarcoma-associated herpesvirus (KSHV)

KSHV is an oncogenic large DNA virus that causes several malignancies, including Kaposi’s sarcoma, primary effusion lymphoma, and aggressive forms of multicentric Castleman disease. KSHV establishes lifelong infection in humans by maintaining a latent/dormant state in B cells and endothelial cells. Activation of the lytic cycle is initiated by expression of the viral Replication and Transcription Activator (RTA), which triggers the temporally coordinated expression of more than 80 viral genes, viral DNA replication, and the production of infectious virions. Using viral mutants, genomics, proteomics, biochemical tools, and advanced imaging techniques, we investigate the molecular mechanisms by which viral and cellular factors regulate KSHV latency and lytic cycle that are crucial for the pathogenesis of KSHV-associated diseases.

Project 1. Molecular basis of the establishment and maintenance of viral latency

Following infection, the chromatin-free 165-kb KSHV genome is delivered into the nucleus of infected cells, where it is maintained as a circular episome and undergoes biphasic chromatinization. During the early stages of infection, the viral genome acquires a transcriptionally permissive chromatin state that supports transient lytic gene expression within the first 24 hours post-infection. Subsequently, the viral episome progressively adopts a repressive heterochromatin structure, mediated by multiple chromatin-regulatory factors that silence viral gene expression and promote latency. Through co-evolution with its host, KSHV has evolved to exploit cellular developmental gene-regulatory pathways, including Polycomb proteins-mediated gene silencing, to establish and maintain persistent infection.

Our research focuses on understanding how the permissive and restrictive chromatins are assembled on the viral genome during infection. We investigate how viral and host factors regulate distinct chromatin states and their functions on the viral genome across diverse cell types and physiological conditions to facilitate viral latency.

Current areas of investigation include:

• Identifying the epigenetic regulators and transcription factors that are recruited to the KSHV genome during infection and elucidating the mechanisms by which they silence viral gene expression to establish and maintain latency.

• Determining how histone modifications associated with viral chromatin regulate viral gene expression and infection outcomes.

• Defining the non-canonical functions of host epigenetic factors (e.g., polycomb proteins) in KSHV infection. Our studies have revealed that several epigenetic regulators can act differently on the viral episome than on the host genome, highlighting previously unrecognized mechanisms of host-virus interaction that remain poorly understood.

Project 2. Viral and cellular regulators of the KSHV lytic cycle

The KSHV lytic cycle is initiated by expression of the viral gene ORF50, which encodes the Replication and Transcription Activator (RTA), the master regulator of lytic reactivation. Upon expression, RTA promotes the lytic program through multiple mechanisms. It functions as a transcription factor that activates the expression of numerous viral and cellular genes required for productive infection, and as an E3 ubiquitin ligase that targets host restriction factors for degradation, thereby creating a cellular environment conducive to viral replication. Lytic reactivation can occur either during primary infection of permissive cell types, such as oral epithelial cells, or in latently infected cells in response to a variety of physiological and environmental stress signals.

Our research aims to define the molecular mechanisms that regulate RTA expression and function during KSHV infection.

Current areas of investigation include:

• Elucidating the epigenetic and transcriptional mechanisms that control RTA induction in different cell types.

• Defining the roles of RTA-regulated host factors in controlling lytic reactivation and productive viral replication.

• Characterizing host proteins that interact with RTA and determining how these interactions influence RTA-mediated viral gene expression and lytic-cycle progression.

• Identifying novel roles of lytic viral factors in promoting the KSHV lytic cycle.

Project 3. Regulation of KSHV de novo infection under hypoxia

The current view that KSHV infection primarily results in the establishment of viral latency is based largely on infection studies performed in cell cultures grown under optimal, stress-free conditions. Although these studies have yielded fundamental insights into the molecular mechanisms of KSHV infection, they fail to recapitulate the physiological stresses that cells encounter during infection in vivo. Understanding how such environmental cues influence viral gene expression and determine the outcome of de novo KSHV infection, latency vs. lytic replication, represents a critical gap in our knowledge of KSHV biology.

One of the most common physiological stress conditions in vivo is hypoxia, which is present in many tissues, including lymphatic microenvironments that support KSHV infection. We recently demonstrated that hypoxia promotes lytic KSHV infection in cell types that ordinarily support only latent infection under normoxic conditions, providing direct evidence that physiological environmental cues can profoundly influence the course of viral infection. Cellular responses to hypoxia are primarily mediated by the hypoxia-inducible factors (HIF-1, HIF-2, and HIF-3), which orchestrate tissue-specific transcriptional programs that regulate adaptation to low oxygen conditions. However, the mechanisms by which HIFs modulate KSHV de novo infection and viral gene expression remain poorly understood. Our research focuses on defining how hypoxia and HIF-dependent pathways regulate KSHV infection and pathogenesis.

Current areas of investigation include:

• Determining how hypoxia influences the establishment of latent and lytic KSHV infections in different cell types.

• Defining the roles of HIF transcription factors in regulating viral gene expression and viral chromatin dynamics.

• Identifying host and viral mechanisms that mediate hypoxia-dependent changes that determine whether KSHV infection progresses toward latency or lytic replication.

• Elucidating the molecular mechanisms by which HIFs and their associated cofactors modulate KSHV latency, lytic cycle, and virus production.

Project 4. Regulation of primary KSHV infection

Here, we investigate the molecular mechanisms that govern the earliest stages of KSHV infection in humans, which occur within the oral cavity. KSHV infects oral epithelial cells, where the virus undergoes lytic replication. Subsequently, the virus sheds into saliva, which facilitates person-to-person transmission while virus transmission from oral epithelial cells to other cells such as B cells help viral dissemination within the host. Despite the critical role of oral KSHV infection in viral transmission and pathogenesis, it remains one of the least understood and most understudied aspects of the viral life cycle.

Our research focuses on how metabolites produced by the oral microbiota influence KSHV infection by activating distinct cell-signaling pathways and stress-response transcription factors in oral epithelial cells that promote viral replication. Oral infection is a crucial step in both the establishment of lifelong KSHV persistence and the saliva-mediated transmission of the virus to new hosts. By elucidating the molecular interactions among oral bacterial metabolites, host stress-response pathways, and KSHV replication, our studies aim to identify novel antiviral targets that can be exploited to prevent infection and limit the spread of this cancer-causing herpesvirus.

Current areas of investigation include:

• Identifying cell-signaling pathways modulated by oral bacteria and their metabolites in oral epithelial cells and determining their roles in facilitating primary KSHV infection.

• Investigating how KSHV dysregulates innate immune response pathways in oral epithelial cells to promote viral spread.

• Defining the epigenetic and transcriptional mechanisms that regulate lytic KSHV infection in oral epithelial cells.