Functional dissection of LANA binding site LBS3/RE (replication element) in viral genome replication and persistence of Kaposi’s sarcoma-associated herpesvirus

Title: Functional dissection of LANA binding site LBS3/RE (replication element) in viral genome replication and persistence of Kaposi’s sarcoma-associated herpesvirus

Abstract:

Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) persists in the nucleus as latent episomes in dividing tumor cells. During the latent phase, viral episomes are tethered to host chromatin and segregate to the daughter cells following replication, which critically relies on the latency-associated nuclear antigen (LANA) protein. Deletion mapping of the latent replication origin revealed two distinctive sequence elements: LANA binding sites (LBS1/2) and a 32-bp long GC-rich sequence upstream of LANA binding site 1 (LBS1), referred to as LBS3/RE (replication element), along with LANA binding sites 2 (LBS2), to be sufficient to support replication. LANA has been shown to specifically bind to LANA binding sites (LBS) within the terminal repeat (TR) region. In this study, we dissected the role of the KSHV replication element, LBS3/RE in viral genome replication, maintenance, and latency establishment using a recombinant KSHV (Bacterial Artificial Chromosome). Our data showed that a single copy of the terminal repeat is insufficient to adequately support replication and episome persistence and TR without the LBS3/RE was further impacted for the episome maintenance. Importantly, recombinants with either a single copy of the TR or LBS3 deleted TR were severely defective for the cell’s production of cell-free virions. Analysis of the chromatin architecture at the TR region and the promoters of latent, immediate-early, and late genes showed proportionately similar ratios of active chromatin (H3K4me3) and repressive (H3K27me3) marks on LANA promoter but was slightly altered on the TR and RTA promoter among these recombinants. We used the SingleMolecule Analysis of Replicated DNA (SMARD) approach to analyze the replication initiation sites among these recombinants and found that viruses with single TR or LBS3 deleted TR initiate replication at the other sites. Together, these results suggest that multiple TRs are required for efficient DNA replication and virion production.

Biography:

Dr. Namrata Gupta studied Biotechnology at the Banaras Hindu University, Varanasi, and graduated as MS in 2006. She then joined the research group of Prof. Anil Kumar Tripathi at the School of Biotechnology, Banaras Hindu University, Varanasi. She worked on characterizing the role of sigma factor in abiotic stress tolerance in a soil bacterium, Azospirillum brasilense. She received her PhD degree in 2013 at the same institution. After completing her doctoral degree, she joined the University of Nevada, Reno School of Medicine as a Post-doctoral researcher supervised by Prof. Subhash C. Verma. The research interests of the laboratory focused on: determining the mechanisms utilized by human herpesvirus 8 (HHV8) in inducing various tumors in humans. HHV8, also called Kaposi’s sarcoma associated herpesvirus (KSHV), is an infectious agent, which establishes latent infection and causes various malignancies in immune compromised individuals. Like other herpesviruses, KSHV encodes for a nuclear protein, Latency Associated Nuclear Antigen (LANA), which is the key protein in tethering the viral genome to the host chromosome. LANA helps in replication of the viral genome and also blocks the immune pathways to hide itself from the host’s immune surveillance system. Her work focused on determining the roles of LANA in replication and the assembly of nucleosomes on the newly synthesized DNA. Her findings show that LANA recruits host cellular nucleosome assembly protein, NAP1L1 to deposit the core histones on the replicated copies of the viral genome. She also worked on how LANA suppresses host immune response to hide from the host immune system. LANA binds to the RFX complex to disrupts the assembly of transcription factors and thus down-regulates the expression of class II major histocompatibility complex (MHCII). Additionally, she worked on understanding the role of LANA in modulation of cellular signaling and persistence during primary infection. These findings were published in many Scopus/SCI peer-reviewed journals like Scientific Reports, Nature Publishing Group, Journal of Virology, Frontiers in Microbiology etc. After her postdoctoral fellowship she obtained the position of an Associate Professor at the Chandigarh University.