Mechanistic differences between nucleic acid chaperone activities of the Gag proteins of Rous sarcoma virus and human immunodeficiency virus type 1 are attributed to the MA domain.
Host cell tRNAs are recruited for use as primers to initiate reverse transcription in retroviruses. Human immunodeficiency virus type 1 (HIV-1) uses tRNA(Lys3) as the replication primer, whereas Rous sarcoma virus (RSV) uses tRNA(Trp). The nucleic acid (NA) chaperone function of the nucleocapsid (NC) domain of HIV-1 Gag is responsible for annealing tRNA(Lys3) to the genomic RNA (gRNA) primer binding site (PBS). Compared to HIV-1, little is known about the chaperone activity of RSV Gag. In this work, using purified RSV Gag containing an N-terminal His tag and a deletion of the majority of the protease domain (H6.Gag.3h), gel shift assays were used to monitor the annealing of tRNA(Trp) to a PBS-containing RSV RNA. Here, we show that similar to HIV-1 Gag lacking the p6 domain (GagΔp6), RSV H6.Gag.3h is a more efficient chaperone on a molar basis than NC; however, in contrast to the HIV-1 system, both RSV H6.Gag.3h and NC have comparable annealing rates at protein saturation. The NC domain of RSV H6.Gag.3h is required for annealing, whereas deletion of the matrix (MA) domain, which stimulates the rate of HIV-1 GagΔp6 annealing, has little effect on RSV H6.Gag.3h chaperone function. Competition assays confirmed that RSV MA binds inositol phosphates (IPs), but in contrast to HIV-1 GagΔp6, IPs do not stimulate RSV H6.Gag.3h chaperone activity unless the MA domain is replaced with HIV-1 MA. We conclude that differences in the MA domains are primarily responsible for mechanistic differences in RSV and HIV-1 Gag NA chaperone function. Importance: Mounting evidence suggests that the Gag polyprotein is responsible for annealing primer tRNAs to the PBS to initiate reverse transcription in retroviruses, but only HIV-1 Gag chaperone activity has been demonstrated in vitro. Understanding RSV Gag's NA chaperone function will allow us to determine whether there is a common mechanism among retroviruses. This report shows for the first time that full-length RSV Gag lacking the protease domain is a highly efficient NA chaperone in vitro, and NC is required for this activity. In contrast to results obtained for HIV-1 Gag, due to the weak nucleic acid binding affinity of the RSV MA domain, inositol phosphates do not regulate RSV Gag-facilitated tRNA annealing despite the fact that they bind to MA. These studies provide insight into the viral regulation of tRNA primer annealing, which is a potential target for antiretroviral therapy.
The Ohio State University, Department of Chemistry and Biochemistry, The Ohio State Biochemistry Program, Center for Retroviral Research, and Center for RNA Biology, Columbus, Ohio, USA.
Tiffiny D
TD
Rye-McCurdy
Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, Pennsylvania, USA Department of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, Pennsylvania, USA.
Shorena
S
Nadaraia-Hoke
Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, Pennsylvania, USA.
Nicole
N
Gudleski-O'Regan
Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, Pennsylvania, USA.
John M
JM
Flanagan
Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, Pennsylvania, USA Department of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, Pennsylvania, USA.
Leslie J
LJ
Parent
The Ohio State University, Department of Chemistry and Biochemistry, The Ohio State Biochemistry Program, Center for Retroviral Research, and Center for RNA Biology, Columbus, Ohio, USA musier@chemistry.ohio-state.edu.
Karin
K
Musier-Forsyth
D024202:Electrophoretic Mobility Shift Assay; D015683:Gene Products, gag / Q000378:metabolism*; D015488:HIV Antigens / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D012364:RNA, Transfer, Trp / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D052736:Rous sarcoma virus / Q000502:physiology*; D014763:Viral Matrix Proteins / Q000378:metabolism*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
D015683:Gene Products, gag; D015488:HIV Antigens; C110263:MA protein, Rous sarcoma virus; D018832:Molecular Chaperones; D010750:Phosphoproteins; D012364:RNA, Transfer, Trp; D012367:RNA, Viral; D014763:Viral Matrix Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1