Roles of Kaposi’s Sarcoma-Associated Herpesvirus Glycoproteins in Viral Entry and Cell Tropism

Kaposi’s sarcoma–associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus responsible for conditions such as Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). It is also associated with KSHV inflammatory cytokine syndrome (KICS). The seroprevalence exhibits significant variability, ranging from approximately 20% to 80% in endemic regions such as sub-Saharan Africa, the Mediterranean Basin, and Xinjiang (China), while generally remaining below 10% in most Western nations, reflecting geographic disparities in disease prevalence. Additionally, region-specific indicators (e.g., a purported association with osteosarcoma in Xinjiang) are still exploratory and necessitate further validation and mechanistic investigation. Despite decades of investigation, two entry checkpoints remained insufficiently characterized. Firstly, the major virion antigen K8.1 is reported to bind to cell surface heparan sulfate and be critical for B-cell infection; however, there is a lack of direct experimental proof regarding whether it mediates attachment, induces or enhances fusion, and whether its role extends to epithelial targets. Secondly, although KSHV mainly enters cells through endocytosis and macropinocytosis, and gB is recognized as the conserved fusogen, there was no definitive evidence showing that low pH directly triggers KSHV gB or that gB itself dictates the endocytosis route. This deficiency is partly attributable to numerous assays utilizing heterologous gB glycoproteins from other γ-herpesviruses, due to the limited fusogenic capability of KSHV gB in standard experimental systems. Clarification of these checkpoints is vital to better understand the molecular mechanisms dictating cell and tissue tropism, as well as to identify potential targets for intervention. This dissertation, therefore, establishes a unifying objective: to define KSHV early entry by exploring (i) a K8.1-dependent attachment checkpoint and (ii) a gB-mediated, low-pH fusion checkpoint, thereby elucidating cell-type tropism and route selection. To achieve this, I employed loss- and gain-of-function approaches, focusing on K8.1 (including a K8.1-knockout virus with genetic complementation). I utilized quantitative attachment and binding assays and conducted fusion tests with gH/gL and gB. Concurrently, we used a dual-fluorescent KSHV to quantify single-particle fusion, engineered a Rhesus monkey rhadinovirus (RRV) chimera carrying KSHV gB, and performed low-pH fusion assays to determine whether gB governs pH-dependent entry. K8.1 was demonstrated to be critical for the infection of multiple epithelial cell lines (HaCaT, HEK-293T, A549), but it was not necessary in lymphatic and blood endothelial cells. I demonstrated that this phenotype results from K8.1 deletion was associated with the attachment phase (decreased virion attachment), and neither K8.1 nor its RRV homolog R8.1 initiated or amplified gH/gL–gB–mediated fusion. In co-culture experiments with B cells, KSHVΔK8.1 efficiently infected BJAB cells but showed impaired infection in MC116 cells, emphasizing a role beyond attachment. Collectively, these findings designate K8.1 as a cell-type–dependent factor, particularly important for epithelial cell infection. At the fusion checkpoint, single-particle imaging revealed that a low endosomatic pH is required for KSHV particle–cell fusion, with the “time-to-fusion” varying by target cell. Inhibiting vesicle acidification abrogated fusion, and replacing RRV gB with KSHV gB rendered RRV infection of B cells overexpressing a viral fusion receptor, EphB3, acidification-sensitive, demonstrating that KSHV gB dictates a low-pH endocytic route. Thus, KSHV gB is a pH-activated fusogen that commits KSHV to fusion from within acidified endosomes. Integrating these findings yields a coherent entry model: K8.1 tunes attachment in a cell-type–specific manner upstream of receptor-driven uptake, and low endosomal pH activates gB to execute membrane fusion. This framework reconciles inconsistencies about K8.1’s function and the pH-dependence of KSHV fusion, explains aspects of tropism, and highlights entry-blocking opportunities at attachment (K8.1), uptake, and fusion (endosome acidification/gB triggering), informing future antiviral and vaccine strategies.