Functional Characterization of the Aminoterminus of SIV Nef

Earl Sawai, UC Davis

Nef is an important factor that is necessary for efficient replication of human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) in peripheral blood mononuclear cells (PBMC). In vivo, Nef has been shown to be a pathogenic factor in both humans and rhesus macaques. Nef has been shown to exert a multitude of functions both on the host cell and the virus by mediating interactions with cellular and viral proteins during replication. Accordingly, the multifunctional nature of this key regulatory protein is likely regulated by conformational changes in protein structure thereby governing the interactions with various cellular proteins. The conformation of Nef is likely to be regulated by post-translational modifications such as myristylation and phosphorylation. The aminoterminus (N-terminus) of the HIV/SIV Nef plays a major role during the viral replication cycle by targeting Nef to the plasma membrane, 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 (e.g., HIV-1 MA, recoverin, c-Abl tyrosine kinase, Src tyrosine kinase, myristylated alanine-rich protein kinase C substrate (MARCKS) and ARF1 GTPase).

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 in in vitro kinase assays (IVKAs). 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 Nterminal kinase (SNTK) domain to a region between amino acids 14-70. Interestingly, three tyrosine residues that are crucial for interaction between the N-terminus of SIV Nef and the Nterminal 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 conformation 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. This represent the first time a kinase has been shown to bind to the N-terminus of SIV Nef. The identification of a putative myristyl switch domain and the linkage of the N-terminal kinase activity to key residues in this region is an important finding for the field. 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 HI-1 uses a variation of a similar strategy for Nef function. By identifying p49 we may be able determine whether it is related to the unidentified kinase that binds to the N-terminus of HIV-1 Nef. By investigating the role of the N-terminus of SIV Nef, we can develop a better understanding of how HIV-1 Nef functions. Accordingly, these studies will facilitate the identification of cellular targets necessary for development of novel inhibitors that block HIV replication.