Interaction of HIV reverse transcriptase with structures mimicking recombination intermediates.
Interactions between human immunodeficiency virus (HIV) reverse transcriptase (RT) and structures mimicking intermediates proposed to occur during recombination (strand transfer) were investigated. One mechanism proposed for strand transfer is strand exchange in which a homologous RNA (acceptor) "invades" a donor RNA.DNA duplex (replication intermediate) on which DNA synthesis is occurring. The acceptor displaces the donor of the duplex and binds to the DNA. During exchange a transient trimeric structure forms. A model structure was designed with a replication intermediate to which an acceptor RNA was bound. The acceptor was bound to the 5'-end of the DNA over a 54-base region, whereas the donor associated with the DNA 3'-end over a 28-base region. The dimeric constituents of the trimer (acceptor RNA.DNA and donor RNA.DNA) were also constructed. The acceptor RNA.DNA formed a branched structure in this case. Results showed that RT could cleave the RNA portion of all the structures examined. Association with junction substrates was less stable as determined by off-rates. On the trimer, RT cleaved both RNAs but showed a clear preference for cleaving the donor RNA region. This preference was accentuated by HIV nucleocapsid protein (NC). Results suggest that during recombination RT generally associates with the donor-RNA portion of the trimer and the acceptor RNA is protected but not immune from cleavage. The partial protection likely allows the acceptor RNA to more easily complete strand exchange and shield this RNA to provide a means to salvage replication if the DNA were to dissociate from the cleaved donor RNA.
Raja
Aarti
A
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park 20742, USA.
DeStefano
Jeffrey J
JJ
D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D014779:Virus Replication
D004279:DNA, Viral; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase