# Project 1 - Herpesvirus Replication: Structural and Biochemical Analysis > **NIH NIH P01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2020 · $266,930 ## Abstract Project 1 Project summary/abstract Our Herpesvirus studies have long been a part of this program project Grant (PPG). Previously we focused on HSV-1 lytic replication but in the last renewal began a transition to KSHV to align our work with our PPG colleagues. Our studies combine high resolution electron microscopy (EM) and other biophysical methods with protein purification and biochemical analysis --a highly productive approach unique to our laboratory. We recently made great strides with our purified HSV-1 replication factors: demonstration of rolling circle replication and a transition in these reactions to DNA network formation, and evidence for origin dependent replication. With the purified KSHV replication factors we are positioned to make major discoveries in the KSHV arena. Our guiding hypothesis is that understanding lytic activation and replication is critical to understanding viral oncogenesis and cancer and further, that scaffolds of filaments formed by KSHV ORF6 protein generate the nuclear replication compartments within which the replication occurs. In Aim I we examine the properties of the purified KSHV replication factors: the polymerase accessory factor, the helicase-promase and the single strand binding protein, ORF6. The work will employ EM, surface plasmon resonance, and biochemical assays. The polarity of assembly of ORF6 along single strand DNA will be determined. Novel properties of the protein which links the helicase and primase together and the polymerase accessory factor will be explored. Genetic mutants will be generated with the Damania group. Assembly of ORF6 into protein scaffolds like those in the nucleus will be examined. In Aim II, rolling circle replication will be explored in vitro using the purified KSHV factors. Products will be compared to replicating DNA isolated from cells undergoing lytic replication at different times. The ability of the in vitro replication system to generate DNA networks typical of late replication products in vivo will be examined. Mutant proteins will be generated with the Damania group and their properties examined. The way in which viral and host proteins organize the latent origin of replication will be examined by EM in collaboration with the Dittmer group. In Aim III, the progress of lytic KSHV replication in cells will be followed at the molecular to cellular levels using light and EM methods, genetic mutations in selected proteins, and new cutting edge EM technologies including cryo-shadowing and a new method (miniSOG) in which proteins are tagged and localized at an EM level in thin sections. Morphology of the mitochondria, cytoskeleton and nucleus will be examined. We will further test our hypothesis that filaments of ORF6 create a scaffold in the nucleus upon which replication takes place. The work is highly integrated with studies in the Damania and Dittmer groups where our results will help to drive their studies. We will continue to devel... ## Key facts - **NIH application ID:** 9986670 - **Project number:** 5P01CA019014-41 - **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL - **Principal Investigator:** JACK D GRIFFITH - **Activity code:** P01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $266,930 - **Award type:** 5 - **Project period:** — → 2022-06-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/9986670 ## Citation > US National Institutes of Health, RePORTER application 9986670, Project 1 - Herpesvirus Replication: Structural and Biochemical Analysis (5P01CA019014-41). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9986670. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*