POLYMORPHISMS IN THE CCR5 GENES OF AFRICAN-GREEN MONKEYS AND MICE IMPLICATE SPECIFIC AMINO-ACIDS IN INFECTIONS BY SIMIAN AND HUMAN IMMUNODEFICIENCY VIRUSES
Se. Kuhmann et al., POLYMORPHISMS IN THE CCR5 GENES OF AFRICAN-GREEN MONKEYS AND MICE IMPLICATE SPECIFIC AMINO-ACIDS IN INFECTIONS BY SIMIAN AND HUMAN IMMUNODEFICIENCY VIRUSES, Journal of virology, 71(11), 1997, pp. 8642-8656
CCR5, a receptor for the CC chemokines RANTES, Mip1 alpha, and Mip1 be
ta, has been identified as a coreceptor for infections by macrophage-t
ropic isolates of human immunodeficiency virus type 1 (HIV-1), To stud
y its structure and function, we isolated cDNA clones of human, Africa
n green monkey (AGM), and NIH/Swiss mouse CCR5s, and we quantitatively
analyzed infections by macrophage-tropic HIV-1 and SIVmac251 after tr
ansfecting human HeLa-CD4 cells with the CCR5 expression vectors, The
AGM and NIH/Swiss mouse CCR5 proteins are 97.7 to 98.3% and 79.8% iden
tical to the human protein, respectively, In addition, we analyzed sit
e-directed mutants and chimeras of these CCR5s. Cell surface expressio
n of CCR5 proteins was monitored by using a specific rabbit antiserum
and by binding the chemokine [I-125]Mip1 beta. Our major results were
as follows. (i) Two distinct AGM CCR5 sequences were reproducibly foun
d in DNA from CV-1 cells, The AGM clone 1 CCR5 protein differs from th
at of clone 2 by two substitutions, Y14N in the amino-terminal extrace
llular region and L352F at the carboxyl terminus, Interestingly, AGM c
lone 1 CCR5 was inactive as a coreceptor for all tested macrophage-tro
pic isolates of HIV-1, whereas AGM clone 2 CCR5 was active. As shown b
y chimera studies and site-directed mutagenesis, the Y14N substitution
in AGM clone 1 CCR5 was solely responsible for blocking HIV-1 infecti
ons, In contrast, both AGM CCR5 clones were active coreceptors for SIV
mac251. Studies of DNA samples from other AGMs indicated frequent addi
tional CCR5 polymorphisms, and we cloned an AGM clone 2 variant with a
Q93R substitution in the extracellular loop 1 from one heterozygote,
This variant CCR5 was active as a coreceptor for SIVmac251 but was onl
y weakly active for macrophage-tropic isolates of HIV-1, In addition,
SIVmac251 appeared to be dependent on the extracellular amino terminus
and loop 2 regions of human CCR5 for maximal infection, Our results s
uggest major differences in the interactions of SIVmac251 and macropha
ge-tropic HIV-1 isolates with 19, N13, and Y14 in the amino terminus;
with Q93 in extracellular loop 1; and with extracellular loop 2 of hum
an CCR5, (ii) The NIH/Swiss mouse CCR5 protein differs at multiple pos
itions from sequences recently reported for other inbred strains of mi
ce. This CCR5 was inactive as a coreceptor for HIV-1 and SIVmac251. St
udies of chimeras that contained different portions of NIH/Swiss mouse
CCR5 substituted into human CCR5, as well as the reciprocal chimeras,
indicated that the amino-terminal region and extracellular loops 1 an
d 2 of human CCR5 contribute to its coreceptor activity for macrophage
-tropic isolates of HIV-1. Specific differences with previous CCR5 chi
mera results occurred because the NIH/Swiss mouse CCR5 contains a uniq
ue substitution corresponding to P183L in extracellular loop 2 that is
nonpermissive for coreceptor activity, We conclude that diverse CCR5
sequences occur in AGMs and mice, that SIVmac251 and macrophage-tropic
HIV-1 isolates interact differently with specific CCR5 amino acids, a
nd that multiple regions of human CCR5 contribute to its coreceptor fu
nctions, In addition, we have identified naturally occurring amino aci
d polymorphisms in three extracellular regions of CCR5 (Y14N, Q93R, an
d P183L) that do not interfere with cell surface expression or Mip1 be
ta binding but prevent infections by macrophage-tropic isolates of HIV
-1, In contrast to previous evidence, these results suggest that CCR5
contains critical sites that are essential for HIV-1 infections.