Specific Roles for Lipids in Virus Fusion and Exit Examples from the Alphaviruses - Fusion of Biological Membranes and Related Problems - page 430

somehow -OH of the cholesterol, the residue was

mutated to a threonine. However, in changing the residue to a threonine
(P226T) the wildtype phenotype was seen, whereas changing it to an alanine
(P226A) produced a srf-3 phenotype for both fusion and infection (Gibbons
and Kielian, 1999). These results suggest that our initial hypothesis was
probably an oversimplification, and that a hydroxyl-containing side chain is
not specifically required at El position 226. Instead, our data in both the
SFV and SIN systems (Lu et al., 1999) indicate that the overall conforma-
tion of the El 226 region is important in modulating the virus cholesterol
dependence.
5.2.1.2. Sequences Involved in the SIN Cholesterol Requirement.
Our data indicate that the region around El residue 226 is involved in the
cholesterol dependence of SFV entry and exit. This region of El is quite
conserved among alphaviruses over a length of 12 residues (Figure 3), but
position 226 itself is not conserved, and can be either proline (SFV and Ross
River virus), valine (western equine encephalitis virus), or alanine (remain-
ing alphaviruses) (Vashishtha et al., 1998). In SIN, the sequence of El
from residue 224-235 is identical to that of SFV except for 226 alanine-lysine-
asparagine (AKN). In order to test the role of this region in SIN sterol-
dependence, various SIN mutants were created (Figure 3), including a single
point mutation changing SIN alanine to serine (SKN), and a triple muta-

substitute for the 3 β

-
tion changing the wt SIN AKN sequence to SGM ( SGM), thus making the

_
sequence identical to that of srf-3 from El 224-235 (Lu et al., 1999). The
SKN mutant is still strongly dependent on cellular cholesterol for its fusion,
infection

_
enhanced infection and fusion with cholesterol-depleted cells compared to
wt SIN, with increases of ~ 100 fold and 250 fold, respectively. Pulse-chase
analysis of virus exit shows comparable kinetics for wt SIN and SGM in

and growth. In contrast, the SGM mutant shows significantly

_
cholesterol-containing cells. In sterol-depleted cells, almost no wt virus is
released, while the SGM mutant shows efficient virus exit. Thus, analogous _

to its role in SFV, the El 226 region acts in the control of the cholesterol
dependence of both SIN entry and exit.
The importance of a specific amino acid or combination of amino acids
in the control of SIN cholesterol dependence was investigated using several
additional mutations in the El 226 region (Lu et al., 1999) (Figure 3). Both
_

the SGN and AGM mutants show increased cholesterol independence, indi-
cating that serine 226

_

and methionine 228 are not specifically required. The

SKM mutant is cholesterol-dependent for infection, indicating that a lysine
at position 227 can abrogate the cholesterol independence of the SG_
mutant. The PGM mutant is also strongly dependent on cholesterol, as
might be predicted from the fact that it has the wt SFV sequence from posi-
tion 224 to 235 on the SIN background. Thus, the overall conclusion from

-

_