Visualizing viral replication of oncogenic human herpesviruses

C.E. Credits: P.A.C.E. CE Florida CE
Speaker

Abstract

Viruses are the causative agents of approximately 12% of human cancers. The most recently discovered herpesvirus, Kaposi’s sarcoma herpesvirus (KSHV) is known to cause three human cancers. Effective antiviral therapeutics are needed for the treatment of KSHV. Viral DNA replication and the KSHV DNA replication proteins are essential to successful viral replication and, thus, appealing therapeutic targets. Our focus is characterizing the functions and molecular interactions of the core KSHV DNA replication proteins in order to develop potential therapeutic strategies against KSHV infection. Herpesviruses encode their own DNA replication machinery. KSHV expresses six DNA replication proteins: DNA polymerase (ORF9), polymerase accessory factor (ORF59), helicase (ORF44), primase (ORF56), primase associated factor (ORF40/41), and single-stranded DNA binding protein (ORF6). The binding of the KSHV polymerase processivity factor, ORF59 (PF-8), to the viral polymerase, ORF9 (Pol-8) promotes continual synthesis of DNA. We visualized ORF59 using negative staining with electron microscopy. We predict head to tail dimers assemble into higher order oligomeric rings composed of four or six monomers, which may function as a sliding clamp during DNA replication. To investigate ORF50 binding in the context of viral DNA replication, we performed in vitro binding assays with purified ORF50 and isolated lytic origin DNA. We visualized purified ORF50 binding to the 2.4kB lytic origin of replication (OriLyt-R) using tungsten metal shadow casting with electron microscopy. We mapped ORF50 binding to OriLyt-R to three distinct regions. Two regions represent novel binding locations. We measured the average area of origin bound ORF50. We predict ORF50 dimerization may stabilize protein-DNA interaction as well as facilitate the binding of additional KSHV DNA replication proteins. Our preliminary finding provides novel insights into the activities of KSHV proteins. Our in-depth molecular study of the core DNA replication proteins will advance the general understanding of KSHV biology.

Learning Objectives:

1. Define the phases of herpesvirus infection

2. Explain high resolution electron microscopy-based approaches to study protein-protein and protein-DNA interactions

3. Identify novel activities of KSHV DNA viral replication proteins.


Show Resources
You May Also Like
SEP 14, 2021 7:00 AM PDT
C.E. CREDITS
SEP 14, 2021 7:00 AM PDT
Date: September 14, 2021 Time: 7am PDT, 10am EDT, 4pm CEST A conventional thermal cycler has long been a commodity product in the lab and end-point PCR techniques can be completed almost wit...
NOV 30, 2021 10:00 AM PST
C.E. CREDITS
NOV 30, 2021 10:00 AM PST
Date: November 30, 2021 Time: 10:00am (PDT), 1:00pm (EDT) The prevalence of thyroid disease worldwide has served as a catalyst for healthcare providers to study various tools and methods to...
NOV 09, 2021 11:00 AM PST
C.E. CREDITS
NOV 09, 2021 11:00 AM PST
Date: November 09, 2021 Time: 11:00am (PDT), 02:00pm (EDT) Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term gro...
DEC 09, 2021 11:00 AM PST
C.E. CREDITS
DEC 09, 2021 11:00 AM PST
Date: December 09, 2021 Time: 11:00am (PDT), 2:00pm (EDT) The burden of antimicrobial resistance (AMR) has been acknowledged worldwide by leading health institutes. Besides the need for new...
OCT 20, 2021 10:00 AM PDT
C.E. CREDITS
OCT 20, 2021 10:00 AM PDT
Date: October 20, 2021 Time:10:00am (PDT), 1:00pm (EDT) As the prevalence of Diabetes continues to rise in many areas across the globe, healthcare providers continue to look for methods that...
SEP 17, 2021 12:00 PM CST
C.E. CREDITS
SEP 17, 2021 12:00 PM CST
Date: September 16, 2021 Time: 9:00pm (PDT), 12:00am (EDT) 3D cellular models like organoids and spheroids offer an opportunity to better understand complex biology in a physiologically rele...
Loading Comments...
Show Resources