Antigens: Influenza Hemagglutinin
and HIV gp120
Erik Mazur(1) and David
© David Marcey, 2001
II. Flu virus hemagglutinin
B. Membrane Fusion
C. Antigenic variability
III. HIV gp120
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.
Enveloped viruses use use spike proteins as molecular mimics of host molecules in order to bind to target cell receptors and gain entry into cells. However, these spikes serve as convenient antigenic surfaces for immune system recognition. Mammalian viruses thus face tremendous selective pressures to continually change their molecular profiles to evade astoundingly responsive immune systems capable of recognizing and destroying viral particles and infected cells. In many cases, natural selection continually yields viral strains that vary considerably in the antigenic regions of spike proteins. These genetic variants may arise and spread through target species periodically, as in the case of annual human flu virus infections. Or, they may be produced during the course of a single infection, as in the HIV variants that arise in the large number of replication cycles that occur over years within a human individual. This tutorial concerns the structure/function/variability of two viral spike antigens, hemagglutinin of the human influenza virus, and glycoprotein 120 (gp120) of the human immunodeficiency virus (HIV).
Stabilization of the hemagglutinin
trimer arises from interactions between
the three major HA2 a-helices in the formation
of a triple-stranded coiled coil . The N-terminal (top) half of the coiled-coil superhelix is tightly
packed with several nonpolar residues in van der
Waals contact around the 3-fold axis . The C-terminus end of the superhelix expands away from the axis with
polar and charged residues interacting electrostatically
B. Membrane Fusion
After binding to sialic acid residues of receptor proteins on host cells, the influenza virus is brought into the cell by endocytosis. The low pH of the resulting endosome, between pH 5 and pH 6, activates a conformational change in the structure of the hemagglutinin molecule. This "fusion-active" state of hemagglutinin triggers the fusion of the viral membrane and the endosome membrane, releasing the viral nucleocapsid into the cytosol of the host cell.
A soluble fragment of hemagglutinin at low pH has been isolated and characterized (Bullough, et al., 1994). This fragment (TBHA2) is prepared at pH 5.0 by digestion with trypsin and thermolysin and contains the first 27 residues of HA1 residues 38-175 of HA2. Although many of the original hemagglutinin residues are lost in this digestion, the major conformational change caused by the acidic environment in the endosome is clear when one compares the conformation of the original HA2 subunit (BHA-right) to that of TBHA2 (left). The subunits are colored in rainbow from amino to carboxy. Residues 55-76 in BHA are recruited to an a-helix in TBHA2 which extends the a-helix of residues 40-55 in BHA 100 Å towards the endosome membrane in TBHA2 (endosome membrane is up). The a-helix of BHA is also moved slightly away from the viral membrane in TBHA2 and the b-sheet/a-helix structure of BHA follows towards the endosome membrane. The functional consequence of the endosomal refolding is a translocation of residues at the end of the a-helix (not shown) to the endosome membrane, where they will fuse with the endosomal membrane. The resulting a-helix (110 Å) is one of the longest known in any protein.
Four major antigenic sites have been located on the hemagglutinin monomer. Site A is a loop that protrudes 8 Å distally from the molecular surface. Site B combines external residues of an a-helix with several residues of the pocket responsible for sialic acid binding. Site C is a bulge 60 Å from the distal tip of the molecule. Amino acids substitutions in Site D are located in two of the b-sheets of the jelly roll (see above). Evidence suggests that single amino acid substitutions within these four regions result in the ability of flu virus to escape immune surveillance and to spread worldwide every year. In addition to minor changes in the antigenic regions, major changes in the antigenic regions have produced the extremely virulent strains that caused the lethal flu pandemics of 1957 and 1968.
The spike protein of Human Immunodeficiency Virus 1 (HIV-1) responsible for binding to host cell receptor molecules is glycoprotein 120 (gp120) , the core of which is shown at left in a complex with a portion of a host cell receptor protein (CD4) and an anti-gp120 antibody Fab fragment (Kwong, et al., 1998). Before looking at the interactions of gp120 with these molecules, let's examine the structure of the core gp120 .
The heart-shaped core of gp120 to your left is oriented pointing downwards to the target membrane, below. The core comprises five a-helices and 25 b-strands that form numerous b-sheets . The b-sheet closest to the host cell membrane, the bridging sheet, connects an inner domain on the left to the outer domain on the right. There are several loops that have high sequence variability, permitting the virus to continually evade immune responses . One of these loops, disordered and therefore not represented in the crystal structure, lies between residues 396 and 410 (spacefilled).
CD4 is bound in a groove of gp120 at the junction of the bridging sheet, inner domain, and outer domain . Although there is not an exact complementarity of surfaces at the interface, CD4 loses 742 Å2 and gp120 loses 802 Å2 of surface accessible to solvent. CD4 residues in contact with gp120 are mostly found in a stretch from 25-64, whereas gp120 residues involved in binding CD4 are distributed over several noncontiguous spans . A crucial interaction between phe43 of CD4 and asp368, glu370, and trp427 of gp120 is observed . These latter residues are conserved in all primate immunodeficiency viruses, and mutation of these residues blocks CD4 binding (reviewed by Kwong, et al., 1998). The gp120 surface at the interface contains numerous hydrophobic residues, a situation that would be thermodynamically unfavorable in a free protein . This suggests that CD4 binding induces significant conformational changes in gp120.
In addition to binding CD4, gp120 binding to the surface chemokine receptor CCR5 is required for HIV infection. Humans carrying variants of CCR5 are resistant to HIV, suggesting that inhibition of CCR5 binding might be an effective way to stop HIV pathogenesis. gp120 affinity for CCR5 in vitro is dramatically enhanced by incubation of gp120 with soluble CD4 (Wu, et al., 1996), demonstrating that the CCR5 binding site on gp120 is formed by conformational changes induced after binding to CD4 (see above). The neutralizing antibody Fab fragment shown bound to gp120 at left overlaps the binding site for CCR5.
HIV uses various forms of molecular trickery to evade immune responses. Most antibodies capable of neutralizing HIV infection access only the surfaces involved in CD4 or CCR5 binding. Most of the envelope protein gp120 surface is hidden from circulating antibodies by glycosylation or by steric exclusion as gp120 (and gp41) form trimers (Wyatt, et al., 1998). Conformational changes also provide means of escape. The conformation of gp120 prior to binding CD4 may display side-chain variability, employing the chameleon-like ability of HIV to change its molecular recognition profile. Also, since the CD4 binding pocket is recessed, antibodies may not see this important antigenic feature.
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Kenyon College, Gambier, Ohio. A first draft of this exhibit was created for
D. Marcey's Molecular Biology class, Biology 63.
2, Department of Biology, California Lutheran University. Address correspondence to this author (see below).