Related Experiment Videos
Amphotropic murine leukemia virus entry is determined by specific combinations of residues from receptor loops 2 and
M D Lundorf1, F S Pedersen, B O'Hara
1Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
Abstract:
Pit2 is the human receptor for amphotropic murine leukemia virus (A-MuLV); the related human protein Pit1 does not support A-MuLV entry. Interestingly, chimeric proteins in which either the N-terminal or the C-terminal part of Pit2 was replaced by the Pit1 sequence all retained A-MuLV receptor function. A possible interpretation of these observations is that Pit1 harbors sequences which can specify A-MuLV receptor function when presented in a protein context other than Pit1, e.g., in Pit1-Pit2 hybrids. We reasoned that such Pit1 sequences might be identified if presented in the Neurospora crassa protein Pho-4. This protein is distantly related to Pit1 and Pit2, predicted to have a similar membrane topology with five extracellular loops, and does not support A-MuLV entry. We show here that introduction of the Pit1-specific loop 2 sequence conferred A-MuLV receptor function upon Pho-4. Therefore, we conclude that (i) a functional A-MuLV receptor can be constructed by combining sequences from two proteins each lacking A-MuLV receptor function and that (ii) a Pit1 sequence can specify A-MuLV receptor function when presented in another protein context than that provided by Pit1 itself. Previous results indicated a role of loop 4 residues in A-MuLV entry, and the presence of a Pit2-specific loop 4 sequence was found here to confer A-MuLV receptor function upon Pho-4. Moreover, the introduction of a Pit1-specific loop 4 sequence, but not of a Pit2-specific loop 4 sequence, abolished the A-MuLV receptor function of a Pho-4 chimera harboring the Pit1-specific loop 2 sequence. Together, these data suggest that residues in both loop 2 and loop 4 play a role in A-MuLV receptor function. A-MuLV is, however, not dependent on the specific Pit2 loop 2 and Pit2 loop 4 sequences for entry; rather, the role played by loops 2 and 4 in A-MuLV entry can be fulfilled by several different combinations of loop 2 and loop 4 sequences. We predict that the residues in loops 2 and 4, identified in this study as specifying A-MuLV receptor function, are to be found among those not conserved among Pho-4, Pit1, and Pit2.
Insights
This study reveals that specific sequences in Pit1 and Pit2 proteins, particularly in loops 2 and 4, can confer amphotropic murine leukemia virus (A-MuLV) receptor function. These functional sequences can be transferred to other proteins, like Pho-4, to enable A-MuLV entry.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Pit2 is the known human receptor for amphotropic murine leukemia virus (A-MuLV).
- Pit1, a related human protein, does not support A-MuLV entry.
- Chimeric proteins of Pit1 and Pit2 suggest Pit1 contains sequences that can confer receptor function in a different protein context.
Purpose of the Study:
- To identify specific sequences within Pit1 and Pit2 that confer amphotropic murine leukemia virus (A-MuLV) receptor function.
- To investigate if these functional sequences can confer A-MuLV receptor activity when introduced into a non-receptor protein (Pho-4).
- To elucidate the roles of extracellular loops 2 and 4 in A-MuLV receptor function.
Main Methods:
- Construction of chimeric proteins between Pit1, Pit2, and Pho-4 (a distantly related protein).
- Introduction of specific Pit1 and Pit2 extracellular loop sequences (loop 2 and loop 4) into the Pho-4 protein.
- Assessing the ability of engineered Pho-4 variants to support A-MuLV entry.
Main Results:
- Introduction of the Pit1-specific loop 2 sequence into Pho-4 conferred A-MuLV receptor function.
- Introduction of the Pit2-specific loop 4 sequence into Pho-4 also conferred A-MuLV receptor function.
- A Pit1-specific loop 4 sequence abolished A-MuLV receptor function in a Pho-4 chimera that already had Pit1-specific loop 2, indicating complex interactions between loops.
Conclusions:
- A functional A-MuLV receptor can be engineered by combining sequences from non-functional proteins.
- Specific Pit1 sequences can confer A-MuLV receptor function when present in a heterologous protein context.
- Both loop 2 and loop 4 residues are critical for A-MuLV receptor function, with flexibility in the specific sequences that can fulfill this role.