Ty3 integrase is required for initiation of reverse transcription.
The integrase (IN) encoded by the Saccharomyces cerevisiae retrovirus-like element Ty3 has features found in retrovirus IN proteins including the catalytic triad, an amino-terminal zinc-binding motif, and a nuclear localization sequence. Mutations in the amino- and carboxyl-terminal domains of Ty3 IN cause reduced accumulation of full-length cDNA in the viruslike particles. We show that the reduction in cDNA is accompanied by reduced amounts of early intermediates such as minus-strand, strong-stop DNA. Expression of a capsid (CA)-IN fusion protein (CA-IN) complemented catalytic site and nuclear localization mutants, but not DNA mutants. However, expression of a fusion of CA, reverse transcriptase (RT), and IN (CA-RT-IN) complemented transposition of catalytic site and nuclear localization signal mutants, increased the amount of cDNA in some of the mutants, and complemented transposition of several mutants to low frequencies. Expression of a CA-RT-IN protein with a Ty3 IN catalytic site mutation did not complement transposition of either a Ty3 catalytic site mutant or a nuclear localization mutant but did increase the amount of cDNA in several mutants and complement at least one of the cDNA mutants for transposition. These in vivo data support a model in which independent IN domains can contribute to reverse transcription and integration. We conclude that during reverse transcription, the Ty3 IN domain interacts closely with the polymerase domain and may even constitute a domain within a heterodimeric RT. These studies also suggest that during integration the IN catalytic site and at least portions of the IN carboxyl-terminal domain act in cis.
Nymark-McMahon
M Henrietta
MH
Department of Biological Chemistry, College of Medicine, University of California, Irvine, California 92697, USA.
Beliakova-Bethell
Nadejda S
NS
Darlix
Jean-Luc
JL
Le Grice
Stuart F J
SF
Sandmeyer
Suzanne B
SB
D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D018626:Retroelements / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
D018076:DNA, Complementary; D004279:DNA, Viral; D018626:Retroelements; D019426:Integrases