Entry of Amphotropic Murine Leukemia Virus Is Influenced by Residues in the Putative Second Extracellular Domain of Its Receptor, Pit2

Abstract

Human cells express distinct but related receptors for the gibbon ape leukemia virus (GALV) and the amphotropic murine leukemia virus (A-MuLV), termed Pit1 and Pit2, respectively. Pit1 is not able to function as a receptor for A-MuLV infection, while Pit2 does not confer susceptibility to GALV. Previous studies of chimeric receptors constructed by interchanging regions of Pit1 and Pit2 failed to clarify the determinants unique to Pit2 which correlate with A-MuLV receptor function. In order to identify which regions of Pit2 are involved in A-MuLV receptor function, we exchanged the putative second and third extracellular domains of Pit1, either individually or together, with the corresponding regions of Pit2. Our functional characterization of these receptors indicates a role for the putative second extracellular domain (domain II) in A-MuLV infection. We further investigated the influence of domain II with respect to A-MuLV receptor function by performing site-specific mutagenesis within this region of Pit2. Many of the mutations had little or no effect on receptor function. However, the substitution of serine for methionine at position 138 (S138M) in a Pit1 chimera containing domain II of Pit2 resulted in a 1,000-fold reduction in A-MuLV receptor function. Additional mutations made within domain II of the nonfunctional S138M mutant restored receptor function to nearly wild-type efficiency. The high degree of tolerance for mutations as well as the compensatory effect of particular substitutions observed within domain II suggests that an element of secondary structure within this region plays a critical role in the interaction of the receptor with A-MuLV.

Amphotropic Murine Leukemia Virus Entry Is Determined by Specific Combinations of Residues from Receptor Loops 2 and 4

“…An interesting observation comes from a comparison of data presented here with recent data published by Leverett and coworkers (12). As mentioned above, the presence of Pit2 loop 2 residues 121 through 141 can confer A-MuLV receptor function upon Pit1 (12); however, the presence of the same Pit2 loop 2 sequence cannot confer this function upon the chimera Pho/P1(4)P2(2) ( Fig. 1 and 3).…”

“…Moreover, comparable chimeras in which the N-or C-terminal parts were derived from HaPit2 only afforded low levels of A-MuLV infection compared to the Pit1-Pit2 and Pit1-RatPit2 hybrids, although HaPit2 is as efficient an A-MuLV receptor as is Pit2 (6). As previously noted by us (13,22) and others (12), these results suggest that A-MuLV receptor function is defined by a combination of both N-and C-terminal receptor determinants, with region A being a C-terminal determinant of A-MuLV receptor function (13,19,22). These observations thus also suggest that Pit1 harbors sequences which may specify A-MuLV receptor function when presented in a protein context different from that of Pit1, e.g., in the Pit1-Pit2 hybrids.…”

“…However, when the middle part of RatPit2 (encompassing loop 3 and the large intracellular domain) was introduced into Pit1 in addition to the N terminus of RatPit2, the resulting chimera (RRG) supported A-MuLV infection at the wild-type level, although the middle part of RatPit2 alone did not confer A-MuLV receptor function upon Pit1 (chimera GRG). Recently, Leverett and coworkers (12) obtained similar results with chimeras between Pit1 and Pit2. Thus, since the Pit2 sequence encompassing loop 3 and the large intracellular domain alone cannot confer A-MuLV receptor function upon Pit1, we suggest that these sequences influence receptor function by modulating the structural organization of the proteins.…”

“…It has previously been suggested by us (13,22) and others (12), based on results obtained on Pit1-Pit2, Pit1-RatPit2, and Pit1-HaPit2 chimeras and on a MusPit1 mutant, that A-MuLV receptor function is defined by a combination of sequences in the N-and C-terminal parts of the receptors. We have here elaborated on this model and suggest that specific combinations of loop 2 and loop 4 sequences determine A-MuLV receptor function.…”

“…The experiment was repeated with similar results. mera Pit1-4 [12]). Moreover, we find that exchange of the C-terminal part of Pit1 loop 2 for the corresponding Pit2 sequence (Pit2 positions 130 through 141) results in a functional A-MuLV receptor (5).…”