Functional Characterization of the Aminoterminus of SIV Nef
Creators: name: University Of California Office Of The President
Publisher: name: University of California Office of the President
publisherIdentifier: https://ror.org/00dmfq477
publisherIdentifierScheme: ROR
Year: 2024

description: Nef is an important factor that is necessary for efficient replication of human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) in white blood cells. In vivo, Nef has been shown to be an important factor for causing disease in both humans and rhesus macaques. Nef has been shown to perform many functions both on the host cell and the virus by interacting with cellular and viral proteins during replication. Therefore, this key regulatory protein is likely regulated by changes in protein structure thereby controling interactions with various cellular proteins. The shape of Nef is likely to be regulated by modifications which add the fatty acid myristate and phosphate. The aminoterminus (N-terminus) of the HIV/SIV Nef plays a major role during the viral replication cycle by targeting Nef to the cell surface, where it mediates early activation, down regulates CD4, MHC-1 and other receptors from the cell surface and facilitates the late phase of viral assembly. Membrane targeting by myristylation is important for Nef function. Careful examination of the N-terminus of SIV Nef reveals a strong sequence homology with the Mason-Pfizer Monkey Virus (MPMV) matrix (MA), which has been reported to contain a myristyl switch mechanism to mediate transport to membrane structures and facilitate virus budding. Myristyl switches have been shown to regulate the conformation and function of several viral and cellular regulatory proteins Moreover, we have found that the N-terminus of SIVmac239 Nef specifically associates with a novel cellular serine-threonine kinase activity that phosphorylates a cellular 49kDa protein of unknown identity. This kinase is different from p21-activated kinase (PAK) that binds to the central core of both HIV-1 and SIV Nef, because it mediates phosphorylation of p49 predominantly on threonine residues. This kinase activity is unique to SIV Nef as HIV-1 Nef does not exhibit this activity. We have mapped the SIV N-terminal kinase (SNTK) domain to a specific region of the protein. Interestingly, three tyrosine residues that are crucial for interaction between the N-terminus of SIV Nef and the N-terminal kinase also correspond to amino acids predicted to be important for the putative myristyl switch. Thus, Nef interacts with two different kinases, SNTK and PAK, via separate domains suggesting that the N-terminus of Nef can interact with different cellular targets and that the shape of this region may influence Nef function. Hypothesis: Interaction between a novel cellular serine-threonine kinase and the N-terminus of SIV Nef in a region that resembles a putative myristyl switch domain is important for viral replication and Nef function. Specific Aims: 1. Determine whether SNTK activation in the putative myristyl switch domain is critical for regulating other Nef functions in vitro (i.e., subcellular localization, CD4 and MHC-I down regulation and enhancing virion infectivity). 2. Identify the cellular p49 protein that is phosphorylated by the novel SIV N-terminal kinase. Significance: This project investigates novel properties that could explain the multifunctional role of Nef in the infected cell. Although the N-terminus of SIV Nef is different from that of HIV-1 Nef, we show that SIV Nef interacts with a novel kinase activity, it is likely that HIV-1 uses a variation of a similar strategy for Nef function. By investigating the role of the N-terminus of SIV Nef, we can develop a better understanding o