Interaction of moloney murine leukemia virus capsid with Ubc9 and PIASy mediates SUMO-1 addition required early in infection.
Yeast two-hybrid screens led to the identification of Ubc9 and PIASy, the E2 and E3 small ubiquitin-like modifier (SUMO)-conjugating enzymes, as proteins interacting with the capsid (CA) protein of the Moloney murine leukemia virus. The binding site in CA for Ubc9 was mapped by deletion and alanine-scanning mutagenesis to a consensus motif for SUMOylation at residues 202 to 220, and the binding site for PIASy was mapped to residues 114 to 176, directly centered on the major homology region. Expression of CA and a tagged SUMO-1 protein resulted in covalent transfer of SUMO-1 to CA in vivo. Mutations of lysine residues to arginines near the Ubc9 binding site and mutations at the PIASy binding site reduced or eliminated CA SUMOylation. Introduction of these mutations into the complete viral genome blocked virus replication. The mutants exhibited no defects in the late stages of viral gene expression or virion assembly. Upon infection, the mutant viruses were able to carry out reverse transcription to synthesize normal levels of linear viral DNA but were unable to produce the circular viral DNAs or integrated provirus normally found in the nucleus. The results suggest that the SUMOylation of CA mediated by an interaction with Ubc9 and PIASy is required for early events of infection, after reverse transcription and before nuclear entry and viral DNA integration.
Yueh
Andrew
A
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Leung
Juliana
J
Bhattacharyya
Subarna
S
Perrone
Lucy A
LA
de los Santos
Kenia
K
Pu
Szy-Yuan
SY
Goff
Stephen P
SP
D001665:Binding Sites; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D000075223:Poly-ADP-Ribose Binding Proteins; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; C485891:PIAS4 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D050828:Protein Inhibitors of Activated STAT; D025842:SUMO-1 Protein; D014157:Transcription Factors; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9