Interaction of HIV Reverse Transcriptase with Structures Mimicking Recombination Intermediates

Abstract

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 RNA⅐DNA 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 RNA⅐DNA and donor RNA⅐DNA) were also constructed. The acceptor RNA⅐DNA 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.Strand transfer is an essential step in retroviral replication and has been shown to occur from terminal regions and internal regions of RNA templates. Minus strand DNA strand transfer is proposed to potentially occur by two different mechanisms (1). In one, DNA being synthesized on an RNA template (referred to as the donor in this case) dissociates from the RNA and binds to a homologous region on a second RNA (referred to as the acceptor) where synthesis continues. A second mechanism proposes that the acceptor RNA actively participates in the displacement of the DNA strand by associating with the DNA and dislodging the donor. Because retroviruses are diploid, the donor and acceptor RNAs would represent the two genome copies in the capsid. Results from previous experiments suggest that viral nucleocapsid protein (NC) 1 stimulates the binding of a complementary RNA acceptor template to the DNA displacing the donor RNA in the process (2). Consistent with the second mechanism above, this implies that strand transfer could proceed through a trimeric complex consisting of the nascent DNA bound to both the RNA template it was being made on (donor) and a second homologous RNA (acceptor). Such a structure has been shown to occur during DNA transfer in vitro (2, 3). Models developed in vivo (4) and in vitro (5, 6) also indicate the likely existence of a trimeric intermediate as one of the mechanisms used for strand ...

Acceptor RNA Cleavage Profile Supports an Invasion Mechanism for HIV-1 Minus Strand Transfer

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Genetic recombination of poliovirus facilitates subversion of host barriers to infection

“…One possibility is that the polymerase interacts with the nascent RNA much more than with the template and the polymerase-nascent RNA complex moves from one template to another (1,23). Another possibility is that a homologous acceptor template adds to form a three-stranded intermediate (44,45). In either case, interactions of the polymerase with the template would need to be enhanced.…”

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