Year of Award
Doctor of Philosophy (PhD)
Other Degree Name/Area of Focus
Biomolecular Structure and Dynamics
Department or School/College
Department of Chemistry and Biochemistry
J. B. Alexander Ross
Kim Hasenkrug, Jack Nunberg, Mark Pershouse
APOBEC3, gGag, glycosylated Gag, MuLV, restriction
University of Montana
APOBEC proteins have evolved in mice and humans as potent innate defences against retroviral infections. APOBEC3G (hA3G) in humans and mouse APOBEC3 (mA3) deaminate cytidine in single-stranded DNA which ultimately results in hypermutation of newly synthesized proviral DNA. Other deaminase-independent mechanisms of inhibition have been identified, such as directly inhibiting reverse transcription. Both HIV and murine leukemia viruses (MuLVs) have evolved mechanisms to evade the action of the APOBEC proteins. HIV encodes the Vif protein which binds to hA3G and facilitates its rapid degradation through the proteasome. The mechanism(s) by which exogenous MuLVs evade mA3 inhibitory activity is unknown.
Exogenous MuLVs encode a glycosylated gag protein (gGag) originating from an alternate CUG start site upstream of the AUG start site of the Gag structural polyproteins. gGag is synthesized to similar amounts as the structural Gag polyprotein in MuLV infected cells but is glycosylated in the endoplasmic reticulum and undergoes distinct proteolytic processing. The function(s) of gGag remain unclear, but eliminating its synthesis through mutation markedly impedes in vivo replication of the virus with very little affect on in vitro replication. Endogenous retroviruses have not been found to express gGag and are tightly controlled by mA3. APOBEC3 proteins are expressed in many tissues in the mouse but are not expressed in most in vitro cell lines. These observations are consistent with a link between gGag expression and the evasion of mA3 by MuLVs.
Studies described herein demonstrate that gGag is protective against both cellular and virion-associated mA3 in vitro and is protective against mA3 in vivo. While there was no direct interaction between mA3 and gGag in an infected cell, gGag and mA3 are localized in the same compartment in the virion and are able to be coprecipitated together from lysed virions. G-to-A hypermutation is not a mechanism used by mA3 to inhibit gGag-negative MuLV replication. Through an affect on reverse transcription, cellular and virion-associated mA3 reduce viral transcripts in MuLV infected cells in a gGag-dependent manner.
Kolokithas, Angelo, "INTERACTIONS BETWEEN THE GLYCOSYLATED GAG PROTEIN OF A MURINE LEUKEMIA VIRUS AND MURINE APOBEC3: NOVEL INSIGHTS INTO HOW A MURINE LEUKEMIA VIRUS COUNTERACTS A RESTRICTION FACTOR" (2011). Graduate Student Theses, Dissertations, & Professional Papers. 585.
© Copyright 2011 Angelo Kolokithas