The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the ␣H and ␣I helices in the thumb subdomain play in specific RNase H cleavage at the 3-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3-end. Further analysis of ␣H mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.The virus-encoded enzyme reverse transcriptase (RT) 1 of human immunodeficiency virus type 1 (HIV-1) and other retroviruses catalyzes the conversion of genomic RNA to a doublestranded DNA replicative intermediate. As minus-strand DNA is synthesized, the RNA template is degraded by the RNase H activity of RT, which cleaves the RNA strand in an RNA-DNA hybrid (Refs. 1 and 2; for reviews, see . This results in the production of many small RNA fragments, any one of which could potentially serve as a primer to initiate synthesis of plus-strand DNA (6). However, a short, purine-rich sequence known as the polypurine tract (PPT) is almost exclusively used as the primer for plus-strand initiation (Refs. 7-12, 61; for a review, see Ref. 6). Why the PPT sequence is selected from all of the other available primers has been the subject of much speculation.One possibility is that the PPT sequence is intrinsically resistant to RNase H degradation and therefore survives as the sole RNA primer available for plus-strand initiation. However, experimental evidence from HIV-1 (13-15) 2 and murine leukemia virus (MuLV) (9, 16, 17) model systems demonstrates that cleavages within the PPT can occur. The MuLV PPT can also be internally cleaved by the isolated RNase H domain from MuLV RT; furthermore, the specificity of cleavage at the 3Ј-end of the PPT is lost when the polymerase domain is removed (18,19). Finally, Escherichia coli RNase H catalyzes cleavages within the MuLV PPT (9,16,17,19) as well as within the HIV-1 PPT. 3A second possibility to explain selection of the PPT primer is related to its unique helical structure (12,20) and the shape and width of the major groove, which is wider than that of other RNA-DNA hybrids (20, 21). These structural factors could cause bi...

Section: Resultsmentioning

confidence: 99%

Residues in the αH and αI Helices of the HIV-1 Reverse Transcriptase Thumb Subdomain Required for the Specificity of RNase H-catalyzed Removal of the Polypurine Tract Primer

Powell¹,

Beard²,

Bębenek³

et al. 1999

“…Although site H was not cleaved in Md1 or Md2, this site was detectably cleaved between the 19th and 20th nucleotides in substrate Md4 (lanes [11][12][13][14][15] and significantly cleaved between the 17th and 18th nucleotides in substrate Md6 (lanes 16 -20). No cleavage was observed at site I in substrates Md1 through Md7 (lanes 1-25), but this site was cleaved when located between the 19th and 20th nucleotides in substrate Md9 and between the 18th and 19th nucleotides in substrate Md10 (lanes [25][26][27][28][29][30][31][32][33][34][35].…”

Section: Sequence and Distance Are Determinants Of Rna 5ј-end-directedmentioning

confidence: 99%

“…Most likely this is because prior studies utilized a limited number of RNAs and thus had very little variation in sequence (21, 29, 32, 38 -43). Also for several studies, the precise mapping of RNA 5Ј-enddirected cleavage sites was often difficult or not required by the experimental design (21,29,32,46). Most recently, RNA 5Ј-end-directed cleavages have been examined with regard to the order of cleavage that occurs on an RNA/DNA hybrid with a recessed RNA 5Ј-end (38 -40).…”

Section: Volume 281 • Number 4 • January 27 2006mentioning

confidence: 99%

“…Third, the polymerase domain can associate with a recessed RNA 5Ј-end, and RNase H can carry out 5Ј-end-directed cleavages that occur ϳ15-20 nucleotides downstream on the RNA strand. While the distance from the recessed RNA 5Ј-end has been characterized as the primary determinant for this mechanism (29), these cleavages have been observed as close as 12-15 nucleotides (30,31) and as far as 21 nucleotides (29,32) from the RNA 5Ј-end. Notably, this range of distances is much broader than the more fixed distance of 17-19 nucleotides that separates the polymerase and RNase H active sites as determined by crystallography studies or footprinting of reverse transcriptase with substrates (33)(34)(35)(36).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Sequence, Distance, and Accessibility Are Determinants of 5′-End-directed Cleavages by Retroviral RNases H

Schultz

Zhang

Champoux

2006

The RNase H activity of reverse transcriptase is essential for retroviral replication. RNA 5-end-directed cleavages represent a form of RNase H activity that is carried out on RNA/DNA hybrids that contain a recessed RNA 5-end. Previously, the distance from the RNA 5-end has been considered the primary determinant for the location of these cleavages. Employing model hybrid substrates and the HIV-1 and Moloney murine leukemia virus reverse transcriptases, we demonstrate that cleavage sites correlate with specific sequences and that the distance from the RNA 5-end determines the extent of cleavage. An alignment of sequences flanking multiple RNA 5-end-directed cleavage sites reveals that both enzymes strongly prefer A or U at the ؉1 position and C or G at the ؊2 position, and additionally for HIV-1, A is disfavored at the ؊4 position. For both enzymes, 5-end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5-end, a distance termed the cleavage window. In examining the importance of accessibility to the RNA 5-end, it was found that the extent of 5-end-directed cleavages observed in substrates containing a free recessed RNA 5-end was most comparable to substrates with a gap of two or three bases between the upstream and downstream RNAs. Together these finding demonstrate that the selection of 5-end-directed cleavage sites by retroviral RNases H results from a combination of nucleotide sequence, permissible distance, and accessibility to the RNA 5-end.In reverse transcription, the single-stranded RNA genome of a retrovirus is transformed into double-stranded DNA by the viral-encoded reverse transcriptase (reviewed in Refs. 1 and 2). This multifunctional enzyme carries out DNA synthesis, strand displacement synthesis, and strand transfer and degrades the RNA portion of RNA/DNA hybrids. The polymerase domain of reverse transcriptase represents the aminoterminal two-thirds of the protein, whereas the RNase H domain comprises the remaining carboxyl-terminal portion. Although the polymerase and RNase H activities are functionally separable, both activities are essential for retroviral replication. The reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) 2 functions as an asymmetric heterodimer composed of p66 and p51 subunits (3), whereas that of Moloney murine leukemia virus (M-MuLV) is a 76-kDa protein that may function as a homodimer or as a monomer (4 -6).RNase H contributes to reverse transcription in three distinct ways (reviewed in Refs. 1, 7, and 8). First, RNase H carries out degradation of the RNA genome both during and after minus-strand DNA synthesis to facilitate plus-strand DNA synthesis and strand transfers. Second, RNase H creates the polypurine tract (PPT) primer from the viral genome. And third, RNase H removes the PPT and tRNA primers used to prime plus-strand and minus-stand DNA synthesis, respectively. Given the vital roles of RNase H in retroviral replication, it is important to understand how RNase H recognizes the RNA/DNA hybri...

“…The third structure, the strand transfer intermediate, contains both the donor and acceptor RNAs bound to DNA. Previous results have indicated that RT is anchored to nucleic acid substrates by a 3Ј recessed DNA terminus or a 5Ј recessed RNA terminus (7)(8)(9)(10)(11)(12)(13). With this in mind, RT would be expected to bind preferentially to the donor RNA of the trimer, because the 3Ј-end of the DNA strand is recessed on the donor RNA.…”

mentioning

confidence: 99%

Interaction of HIV Reverse Transcriptase with Structures Mimicking Recombination Intermediates

Raja¹,

DeStefano

2003

Self Cite

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 ...