Background
Kaposi Sarcoma (KS) is the most common cancer in people living with HIV (PLWH), particularly in countries where Kaposi Sarcoma herpesvirus (KSHV) is endemic. Here, the overall survival of KS patients has changed little over the past twenty years despite increased access to cART. We had previously published the clinical data associated with a prospective study of HIV+ KS patients in Malawi. Here, we describe KSHV stains of additional tumor biopsies and plasma samples from Sub-Saharan Africa. (1) An analysis of currently available (n > 150) full-length viral genomes identifies two different virus lineages that co-circulate in KSHV endemic regions today. The KSHV strains circulating in PWLH today differ from the reference sequence in GenBank and those of common laboratory strains, which originated in the 1990s in the US and Europe. (2) Unbiased long-read sequencing of viral and cellular DNA resolved the terminal repeat structures of the circular KSHV plasmids persisting in several lymphoma cell lines. Also, it yielded the complete human genome for these.
Methods
Library preparation and sequencing were performed using a fully automated approach on the Ion Torrent Genexus Integrated Sequencer (Life Technologies) with a 24-hour turnaround. This used custom primers to enrich KSHV. Targeted short-read sequencing accuracy was validated by PacBio-based long-read sequencing to resolve repeats. Analysis was conducted in CLC Genomics Workbench v23.0.2 (Qiagen Inc.) and BEAST. Structures of wild-type and variant proteins were predicted using AlphaFold2.
Results
The average coverage for the KSHV genomes isolated from FFPE biopsies was 12,206-fold. We identified over one thousand non-synonymous single nucleotide variants (SNV) relative to the current reference strain NC_009333. Most were private, i.e., specific to one individual’s virus. Within each of the two lineages, KSHV continues to evolve over time and across national borders by genetic drift and recombination. Analyses of shared SNVs by AlphaFold2 predicted that some SNVs change the conformation of viral proteins essential in replication, pathogenesis, and infection.
Conclusions
These findings may help our understanding of KSHV evolution and inform KSHV vaccine design. It fills the gap of fully sequenced KSHV genomes of 300 (as compared to 6,000,000 for SARS-CoV-2). Integrating structural AI with conventional phylogenetic analyses pinpoints evolutionary pivots as the target for vaccine and drug susceptibility design.