# Synthetic Genomics Approach to Assemble Infectious Clones of KSHV

> **NIH NIH R03** · JOHNS HOPKINS UNIVERSITY · 2020 · $89,551

## Abstract

The DNA genomes of herpesviruses range in size from 120 kb to 240 kb and hence, have a large coding
capacity in some cases in excess of 100 gene products. For years, genetic manipulation of these genomes
was feasible for only a subset of these viruses. Subsequently, many of the herpesvirus genomes were cloned
into BAC plasmids, which significantly advanced the technologies of genome engineering in an E.coli host and
the successful reconstitution of infectious virus in the appropriate host cell. In this application, we propose a
transformational approach, which is to use synthetic biology to build wild-type clones of the Kaposi's sarcoma-
associated herpesvirus (KSHV) genome and demonstrate the reconstitution of infectivity of these assembled
genomes. The successful outcome of this synthetic genomics approach will significantly advance the ability to
clone, assemble and engineer this important virus in a more high-throughput manner.
Specific Aim 1: Use synthetic genomics methods to clone and assemble KSHV genomes in yeast. In
this aim, we will clone the KSHV genomes from two strains, BCBL-1 and JSC-1, using synthetic genomics
methods. These genomes will be deconstructed into 11 parts, which can be modified separate of each other
and then reassembled using yeast homologous recombination. Our approach will be based on advances made
in this field by members of the J. Craig Venter Institute (JCVI) team that created the first synthetic microbe. In a
collaborative effort, we have already assembled an infectious clone of herpes simplex virus type-1 (HSV-1)
using these methods and are close to completion of an Epstein-Barr virus (EBV) Akata genome. We will use
these new and powerful synthetic genomics methods to assemble complete genomes of KSHV in yeast.
Specific Aim 2: Reconstitute biological activity of the assembled KSHV genomes in mammalian cells.
The goal in this aim will be to recover infectious virus after introduction of assembled herpesvirus genomes into
mammalian cells. KSHV assembled genomes will be transfected/electroporated endothelial cells (TIME -
telomerase-immortalized microvascular endothelial cells) and BJAB cells. Cell lines that harbor the KSHV
episome, following drug selection, will be induced for lytic virus production. Biological activity will be measured
using latency antigen (LANA) staining to measure establishment of latency as well as spindle cell conversion of
endothelial cells. Virus reactivation following lytic activation will be determined using quantitative PCR to
measure viral genomes, lytic gene expression as well as TIME GFP titers.
Our singular goal is to use the combined and complementary expertise of the JHU and JCVI laboratories to
demonstrate we can assemble complete genomes of herpesviruses from the individual parts in an efficient
process with high fidelity and stability. If successful, this would provide a new powerful platform to clone and
manipulate these viruses to facilitate the study of their biology. This...

## Key facts

- **NIH application ID:** 9930053
- **Project number:** 5R03AI146632-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** PRASHANT J DESAI
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $89,551
- **Award type:** 5
- **Project period:** 2019-05-15 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9930053

## Citation

> US National Institutes of Health, RePORTER application 9930053, Synthetic Genomics Approach to Assemble Infectious Clones of KSHV (5R03AI146632-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9930053. Licensed CC0.

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