Board 23: Characterization of the Effects of IP6 on Jaagsiekte Sheep Retrovirus (JSRV) Assembly

Student Scientist: Myles Steed ’23
Research Mentors: Clifton L. Ricaña, Savannah Brancato, Volker Vogt, and Robert A. Dick (Department of Molecular Biology & Genetics, MBG-REU program, Cornell University)

Jaagsiekte Sheep Retrovirus (JSRV) is a virus that is known to cause lung cancer in sheep. JSRV’s genetic composition is similar to other viruses such as HIV, so it was thought that the structure of JSRV would be related as well. Furthermore, a chemical made naturally in cells known as inositol hexaphosphate (IP6) has been found to be needed for forming the structure of HIV and contribute to further disease, however, little is known about IP6’s effect on JSRV. To study these questions about JSRV we utilized microscopy and biochemical lab techniques in the hopes to identify viral targets for treatment.

Jaagsiekte Sheep Retrovirus (JSRV) is a retrovirus that causes ovine pulmonary adenocarcinoma. As for other retroviruses, the structural protein of JSRV is Gag, which is a multi-domain protein. In assembly of a virus particle, several thousand molecules of Gag come together to form the immature lattice. During the maturation process of JSRV, the viral protease cleaves the Gag on the interior side of the viral membrane releasing the CA subdomain, which is necessary for formation of an infectious virus. The lattice structures formed by Gag and by the released CA domain differ from each other, but are known for other retroviruses such as HIV-1, though less is known for JSRV. To better understand the transition between the two distinct lattices, we wish to determine the structure of the immature lattice of JSRV, which can be formed in vitro from a truncated Gag protein including CA and the adjacent NC domain. And we want to understand how cofactors like inositol hexaphosphate (IP6) affect maturation of the CA lattice. We used the following approaches to address these two unknowns. To study the JSRV CA-NC lattice assembly in vitro, we introduced an expression plasmid encoding CA-NC into E. coli, purify the translated immature CA-NC protein, incubate the protein under conditions where it will form immature virus-like particles, and then solve the structure of the lattice utilizing single particle cryo-electron microscopy. To study the role of IP6 on JSRV assembly, budding, and maturation in vivo, we transfected a plasmid expressing Gag into both wild-type and IP6-deficient mammalian cells and measured the release of virus particles from cells and the level of maturation by immunoblotting. Results from these experiments will allow us to identify key targets for antiretroviral development.