Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities

Wöhrl, Birgitta M.; Volkmann, Silke; Moelling, Karin

doi:10.1016/0022-2836(91)90119-q

Cited by 44 publications

(62 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The generation of primary cuts at a pause site during synthesis was in agreement with the findings of Suo and Johnson (50). Other studies also identified 3Ј enddirected secondary cuts in the absence of synthesis (31,(51)(52)(53)(54)(55).…”

supporting

confidence: 88%

Mechanisms That Prevent Template Inactivation by HIV-1 Reverse Transcriptase RNase H Cleavages

Purohit

Roques

Kim

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

The RNase H activity of human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) cleaves the viral genome concomitant with minus strand synthesis. We previously analyzed RT-mediated pausing and RNase H cleavage on a hairpincontaining RNA template system and reported that RT generated 3 end-directed primary and secondary cuts while paused at the base of the hairpin during synthesis. Here, we report that all of the prominent cleavage products observed during primer extension on this template correlated with pause induced cuts. Products that persisted throughout the reaction corresponded to secondary cuts, about eight nucleotides in from the DNA primer terminus. This distance allows little overlap of intact template with the primer terminus. We considered whether secondary cuts could inactivate further synthesis by promoting dissociation of the primer from the template. As anticipated, 3 end-directed secondary cuts decreased primer extendibility. This provides a plausible mechanism to explain the persistence of secondary cut products in our hairpin template system. Improving the efficiency of synthesis by increasing the concentration of dNTPs or addition of nucleocapsid protein (NC) reduced pausing and the generation of pause related secondary cuts on this template. Further studies reveal that 3 end-directed primary and secondary cleavages were also generated when synthesis was stalled by the presence of 3-azido-3-deoxythymidine at the primer terminus, possibly contributing to 3-azido-3-deoxythymidine inhibition. Considered together, the data reveal a role for NC and other factors that enhance DNA synthesis in the prevention of RNase H cleavages that could be detrimental to viral replication. Human immunodeficiency virus, type 1 (HIV-1)3 reverse transcriptase (RT) is the virally encoded enzyme responsible for conversion of the single-stranded RNA genome of HIV-1 to a double-stranded DNA form that can be integrated into the host genome (1). RT can utilize both RNA and DNA as a template for DNA synthesis. In addition, RT possesses RNase H activity, meaning that it can cleave RNA within a RNA:DNA hybrid. Both the polymerase and RNase H activities of RT are essential for the reverse transcription of the viral genome.RT is a p66/p51 heterodimer with both the polymerase and RNase H activities residing in the p66 subunit. When RT binds the primer terminus within the polymerase active site, the RNase H active site is positioned 18 nt upstream of the primer terminus (2-5). Concomitant with synthesis of the first, or minus DNA strand, the polymerizing RT generates cuts on the RNA template ϳ18 nt upstream, designated "polymerase-dependent" cuts. However, the polymerase and RNase H activities of RT are uncoupled and cuts occur less frequently than nucleotide incorporation (6). Studies in vitro indicate that RNase H cleavage by the polymerizing RT is not sufficient to fully degrade the RNA template and some fragments of RNA are left bound to the cDNA during minus strand synthesis (7). It is believed that these fr...

show abstract

supporting

confidence: 88%

Mechanisms That Prevent Template Inactivation by HIV-1 Reverse Transcriptase RNase H Cleavages

Purohit

Roques

Kim

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In addition to the carboxylates, there are three residues (Ser, His, and Asn) that are conserved in all retroviral and bacterial RNase H sequences (156). Although these side chains are located at the RNase H active site, mutations at these positions have much less effect than mutations in the carboxylates, and it has been suggested that these residues may play some role in substrate binding (159)(160)(161). Indeed, model building of an RNA-DNA substrate suggests that His539 may interact with the phosphate backbone (J Jager, TA Steitz, unpublished) .…”

Section: Ribonuclease Hmentioning

confidence: 99%

Function and Structure Relationships in DNA Polymerases

Joyce¹

1994

Annual Review of Biochemistry

134

View full text Add to dashboard Cite

“…Synthesis of the second DNA strand necessitates complete removal of the original RNA (7,8). The RNA genome is cut into small segments during and after synthesis of the RNA͞DNA hybrid (9)(10)(11). Studies show that two different modes of RNase H activity are important for the removal of the RNA (5,12,13).…”

mentioning

confidence: 99%

“…The first mode is carried out by the same RT performing DNA synthesis. This is the polymerization-dependent mode of cleavage (9)(10)(11)(14)(15)(16)(17). We showed that the HIV-1 RT synthesizing DNA leaves RNA oligomers in its wake, many still bound to the extended DNA primer (17,18).…”

mentioning

confidence: 99%

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

Wisniewski

Balakrishnan

Palaniappan

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5 end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5 end of the RNA to make a secondary cut creating two products 8 -9 nt long. RT then binds the new 5 end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3 end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.H IV is the causative agent of AIDS (1). To establish an infection, HIV must integrate a double-stranded viral DNA into the host chromosome (2). The virus-encoded reverse transcriptase (RT) converts the single-stranded viral RNA genome into that double-stranded DNA. RT uses several different catalytic activities in this process. The polymerase function catalyzes DNA synthesis on RNA and DNA templates (3). RT also has an RNase H active site that cleaves RNA when annealed to DNA. RNase H activity is required for a number of steps in viral replication, including formation of primers and primer strand transfers (4). An important role for the RNase H is the complete removal of the original genomic RNA during the synthesis of the double-stranded DNA (5, 6).The genomic viral RNA first is converted to an RNA͞DNA hybrid and then to double-stranded DNA. Synthesis of the second DNA strand necessitates complete removal of the original RNA (7,8). The RNA genome is cut into small segments during and after synthesis of the RNA͞DNA hybrid (9-11). Studies show that two different modes of RNase H activity are important for the removal of the RNA (5, 12, 13). The first mode is carried out by the same RT performing DNA synthesis. This is the polymerization-dependent mode of cleavage (9)(10)(11)(14)(15)(16)(17). We showed that the HIV-1 RT synthesizing DNA leaves RNA oligomers in its wake, many still bound to the extended DNA primer (17, 18). These residual RNAs occur because cleavages of the RNA template happen less frequently than nucleotide addition. Kati et al. (19) examined the rates of both catalytic activities of RT and found that the polymerization rate is 7-10 times faster than the RNase H rate. This result demonstrates that the two activities are uncoupled, such that several nucleotides are added in the time required fo...

show abstract

Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities

Cited by 44 publications

References 30 publications

Mechanisms That Prevent Template Inactivation by HIV-1 Reverse Transcriptase RNase H Cleavages

Mechanisms That Prevent Template Inactivation by HIV-1 Reverse Transcriptase RNase H Cleavages

Function and Structure Relationships in DNA Polymerases

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

Contact Info

Product

Resources

About