II. Structure of the Core Domain
III. CD4 Downregulation
IV. Signal Transduction Interference
Note: This exhibit is best viewed if the cue buttons () are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.
Although HIV-1 Nef was originally named "negative factor," it has been shown to have a positive role in viral replication and pathogenesis. Nef is a viral protein that interacts with host cell signal transduction proteins to provide for long term survival of infected T cells and for destruction of non-infected T cells (by inducing apoptosis). Nef also advances the endocytosis and degradation of cell surface proteins, including CD4 and MHC proteins (CD4 is an integral membrane protein that functions in T-cell activation, and is the receptor for the HIV virus). This action possibly impairs cytotoxic T cell function, thereby helping the virus to evade the host immune response (e.g., Schwartz, et al., 1996). The multifunctional protein thus helps the virus maintain high viral loads and overcome host immune defenses, contributing to the progression of AIDS. Not surprisingly, persons infected with HIV-1 strains that have deletions of the Nef gene develop AIDS symptoms much more slowly than those infected with standard HIV strains. Nef may therefore be a valuable target for pharmaceutical intevention in AIDS progression.
Nef is a 27kD, N-terminal myristoylated accessory protein involved in post integration infection. Nef is found in the viral particle (See Figure 1) and is one of the first proteins to be detected after invasion of the host cell. Only the structure of core domain (at left) has been solved.
II. Structure of the Core Domain
The HIV-1 Nef core domain structure resembles that of the helix-turn-helix (HTH) family of proteins involved in binding DNA (see exhibits on 434 repressor and cro). Three a-helices and a five-stranded antiparallel b sheet are observed in the core domain. The polyproline helix, containing four prolines is a prominent feature . The core of the molecule is hydrophobic , providing numerous hydrophobic bonds to maintain structure. Hydrophobic packing of residues of a -helices 1 and 2 is observed , and a-helix 3 forms hydrophobic interactions with beta strands b1 and b2 . HIV-1 Nef proteins from several strains have been compared, revealing five conserved blocks of residues: Block A (residues 64-90), block B (residues 106-114), block C (residues 130-148), block D (residues 179-190) and the polypurine tract (residues 91-96) . The block containing residues 150 to 180, containing glycine and valine, and the N-terminus are poorly conserved .
III. CD4 Downregulation
HIV-1 Nef is involved in CD4 downregulation. Mutations in the a- or polyproline helices of the A block, have no effect on the downregulation of CD4 . However, residues 60-71, 96-144 and 177-190 of HIV-1 Nef are critical for CD4 downregulation and subsequent viral replication . The Nef/CD4 binding site overlaps the second a helix and links a -helices 1 and 2 of the Nef molecule . This site contains a hydrophobic patch centered at the HIV protease site (trp57, leu58, and glu59) . A negatively charged glutamic acid is also thought to interact with the cytoplasmic tail of the CD4 molecule . The myristoylated signal is required for downregulation of CD4, but the remainder of the amino terminal is not essential.
HIV-1 Nef binds tightly to cellular protein kinases, resulting in interference of normal cellular signaling processes and enhanced infectivity of HIV-1 virons. The Src homology region 3 (SH3) domain in Fyn cellular tyrosine kinase protein is involved in signal transduction. The interaction of the Fyn R961 Mutant SH3 domain with Nef occurs at a type II, left-handed polyproline (PxxP) helix. In the Fyn mutant, arginine-96 is replaced by isoleucine. The HIV-1 Nef core protein (residues 71-203) complexed with R961 Fyn Mutant SH3 domain (residues 85-141) is an asymmetric dimer . The SH3 domain comprises a b barrel structure with numerous conserved hydrophobic residues whose sidechains pack in the barrel's core .
Hydrophobic bonds, hydrogen bonds, and ionic bonds provide interactions between the SH3 domain and the polyproline helix of Nef. Prolines 72 and 75 of the polyproline type II helix of Nef interact with conserved sidechains of the Fyn SH3 domain . Arg77 of the Nef protein stacks against trp119 and forms a salt bridge with asp100 . Hydrogen bonding occurs between the nitrogen atom of arg71 of Nef and the hydroxyl group of tyr137 in the SH3 domain . Pro78 of the Nef protein packs against Nef tyr120 and plays a structural role in SH3 recognition by causing a bend in the loop connecting the PPII helix to the second a-helix . Pro78 does not directly bind to the SH3 domain. Val74 of the Nef protein interacts with several SH3 domain tyrosine residues .
The RT loop of the Fyn SH3 domain, named for its critical arginine and threonine residues, is also important in the interaction with Nef. On the surface of Nef, two antiparallel a-helices, separated by 10.5 Å and a 70o angle, form a hydrophobic pocket. Isoleucine 96 of the RT loop is inserted between in this pocket .
HIV-1 Nef influences viral infectivity in multiple ways. CD4 (and MHC) downregulation and interference with normal signal transduction pathways are both features of Nef action. These roles are reflected in different functions determined by distinct structural features of the Nef protein.
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Lee, C., K. Saksela, U. R. Mirza, B. T. Chait, and J. Kuriyan. "Crystal Structure of the Conserved Core of HIV-1 Nef Complexed with a SCR Family SH3 Domain." Cell. 1996. 16: 931-42.
Grzesiek, S., S. J. Stahl, P. T. Wingfield, and A. Bax. "The CD4 Determinant for Downregulation by HIV-1 Nef Directly Binds to Mapping of the Nef Binding Surface by NMR." Biochemistry. 1996. 35: 10256-10261.
Grzesiek, S., A. Bax, G. M. Clore, A. M. Gronenborn, J. Hu, J. Kaufman, I. Palmer, S. J. Stahl, and P. T. Wingfield. "The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase."Nature Structural Biology. 1996. 3(4): 340-45.
Hua, J. W. Blair, R. Truant, and B. R. Cullen. "Identification of Regions in HIV-1 Nef Required for Efficient Downregulation of Cell Surface CD4." Virology. 1997. 231: 231-238.
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S LeGall, F Lemonnier, and JM Heard. Endocytosis of major histocompatability
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1, Kenyon College, Gambier,
Ohio. A first draft of this exhibit was created for D. Marcey's Molecular
Biology class, Biology 63.
2, California Lutheran University. Address correspondence to this author (see below).
3, California Lutheran University