Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H.

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

doi:10.1016/s0021-9258(18)53827-8

Cited by 34 publications

(16 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Analysis of RNase H CleaVage Products by Denaturing Gel Electrophoresis. The RNase H substrate [ 35 S]poly(rA)‚ poly(dT) was prepared using E. coli RNA polymerase (Volkmann et al, 1993). In a total volume of 300 µL, 20 µg of poly(dT) was incubated for 1 h at 37 °C with 50 units of E. coli RNA polymerase, 50 nM ATP, and 100 µCi of [R-35 S]ATP in 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM KCl, 5 mM MgCl 2 , 5 mM DTT, and 1 mM MnCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Characterization of the p68/p58 Heterodimer of Human Immunodeficiency Virus Type 2 Reverse Transcriptase

Fan¹,

Rank²,

Poppe³

et al. 1996

Biochemistry

View full text Add to dashboard Cite

Recently we demonstrated that the p58 subunit of p68/p58 HIV-2 reverse transcriptase (RT) heterodimer, produced by processing of p68/p68 homodimer with recombinant HIV-2 protease, terminates at Met484 [Fan, N., et al. (1995) J. Biol. Chem. 270, 13573-13579]. Here we describe purification and characterization of the p68/p58 heterodimer of recombinant HIV-2 RT. It exhibited both RT and RNase H activities, obeyed Michaelis-Menten kinetics, and was competitively inhibited by the DNA chain terminator ddTTP (Ki[app] = 305 +/- 20 nM). The HIV-2 RT-associated RNase H exhibited a marked preference for RNA hydrolysis from a HIV-1 gag-based heteropolymeric RNA/DNA hybrid in the presence of either Mg2+ or Mn2+, compared to the [3H]poly(rA).poly(dT) or [3H]poly(rG).poly(dC) homopolymeric substrates. Relative to HIV-1 RT, the RNase H activity of HIV-2 RT was only 5% toward the [3H]poly(rA).poly(dT) in the presence of Mg2+. The size distribution of products generated from [3H]poly(rA).poly(dT) by HIV-2 RT-associated RNase H was markedly distinct from that of HIV-1 RT in the presence of Mg2+ or Mn2+. The p68/p58 HIV-2 RT heterodimer, produced by specific cleavage using HIV-2 protease, should be useful for inhibition and biophysical studies aimed at discovering and designing drugs directed toward HIV-2.

show abstract

Section: Methodsmentioning

confidence: 99%

Characterization of the p68/p58 Heterodimer of Human Immunodeficiency Virus Type 2 Reverse Transcriptase

Fan¹,

Rank²,

Poppe³

et al. 1996

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Positions D460, P468, L469, T470, H483, I506, K512, S519, Q524, K530, Q547, A554 and K558 (having previously been reported to be associated with ART, showing an increased prevalence in treatmentexperienced patients or affecting the functionality of enzymatic processes [13,[19][20][21][22][23]), did not meet our criteria for statistical significance (Table 2). In treatmentexperienced patients, there was an increased prevalence of mutations at positions K527, K530 and Q547 in our analyses, as was found in previous studies; however, position I506 did not show an increased prevalence as it had before [13].…”

Section: Discussionmentioning

confidence: 96%

“…An RT mutation profile analysis of the Stanford database [28] also showed an increased prevalence of R284K, N348I and K451R in treatment-experienced patients (P<0.001 for each), whereas the R284K frequencies were 1.5% (65/4,211) versus 3.7% a Previously reported mutations that might confer or increase resistance to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors [6,9,[16][17][18][19][20][21][22][23]. b Previously reported mutations associated with a decreased prevalence as a result of treatment [13].…”

Section: Discussionmentioning

confidence: 99%

“…Mutations at positions Q475, N494, H539 and D549 have been previously reported to affect RNase H enzymatic function [17][18][19][20][21]. In our analysis, however, a high conservation at these positions in both groups (in the treatment-naive group, Q475, N494 and D549 were 100% conserved and H539 was 99% conserved and in the treatment-experienced group, all four were 100% conserved; Table 2) indicates that although mutations at these positions affect enzymatic functions in vitro, their occurrence in vivo is highly infrequent.…”

Section: Discussionmentioning

confidence: 99%

“…In vitro experiments have shown that mutations at highly conserved AA positions within the RNase H region (D475E, N494D, H539N and D549N) can alter the RNase H enzymatic activity (processive activity and binding affinity), thereby affecting the replication cycle of HIV-1 [17][18][19][20]. In vitro experimental data also suggest that the Q509L mutation in the RNase H region, when combined with other mutations (particularly A371V and TAMs in the polymerase region), increases resistance to AZT, lamivudine (3TC) and abacavir [21].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Mutations in the thumb–connection and RNase H domain of HIV type-1 reverse transcriptase of antiretroviral treatment-experienced patients

et al. 2009

View full text Add to dashboard Cite

Background Antiretroviral therapy that targets HIV type-1 (HIV-1) reverse transcriptase (RT) can be linked to mutations in the thumb–connection (amino acids [AA] 241–426) and RNase H (AA 427–560) domains, which could affect drug resistance. Methods Genotypical and statistical analyses were performed on HIV-1 RT from 100 antiretroviral treatment-naive and 248 antiretroviral treatment-experienced patients, the majority of whom were infected with HIV-1 subtype B. The RT region was analysed in three parts: the polymerase (AA 1–240), thumb–connection (AA 241–426) and RNase H (AA 427–560) domains. Results The polymerase domain had statistically significant changes between the two groups at 24 AA positions that are known resistance sites. Within the thumb–connection domain, R284 and N348 had statistically significant changes between the groups ( P=0.007 and P≤0.001, respectively). In treatment-experienced patients, 17.3% had R284K, whereas 24.5% had N348I substitutions. Both R284 and N348 were 100% conserved in treatment-naive patients. Within the RNase H domain, only K451 showed a statistically significant change ( P≤0.001), with K451R present in 11% of treatment-experienced patients but remaining 100% conserved among treatment-naive patients. Conclusions RT mutations at three positions outside of the polymerase region were associated with antiretroviral therapy: R284K, N348I and K451R. Both R284K and K451R interact with the phosphate backbone of the template or primer in HIV-1 RT crystal structures and could potentially influence the positioning of the primer strand, thus affecting polymerization, the efficiency of nucleoside reverse transcriptase inhibitor excision and/or RNase H activity.

show abstract

RibonucleaseH

Goedken

Marqusee

2002

Encyclopedia of Molecular Biology

View full text Add to dashboard Cite

Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H.

Cited by 34 publications

References 47 publications

Characterization of the p68/p58 Heterodimer of Human Immunodeficiency Virus Type 2 Reverse Transcriptase

Characterization of the p68/p58 Heterodimer of Human Immunodeficiency Virus Type 2 Reverse Transcriptase

Mutations in the thumb–connection and RNase H domain of HIV type-1 reverse transcriptase of antiretroviral treatment-experienced patients

RibonucleaseH

Contact Info

Product

Resources

About