Human immunodeficiency virus type‐1 reverse transcriptase and ribonuclease h as substrates of the viral protease

Tomasselli, Alfredo G.; Sarcich, Jean L.; Barrett, L; Reardon, Ilene M.; Howe, W. Jeffrey; Evans, David B.; Sharma, Satish Kumar; Heinrikson, Robert L.

doi:10.1002/pro.5560021216

Cited by 37 publications

(47 citation statements)

References 41 publications

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“…2 and 6 and Table 2, we conclude that the two subunits in p66/p66 homodimer are not symmetrically related, presumably as a result of partial unfolding of the C-terminal p15 RNase H domain in one of the p66 protomers. This conclusion is consistent with our recent comparative studies on the susceptibility of ~661~66 homodimer and the active p15 RNase H domain to digestion by HIV-l protease [19]. The origin of conformational differences in the two p66 subunits, as elaborated by peaks 1 and 2 of Fig.…”

Section: Sk Sharma Et Al Ifebs Letters 343 (1994) 125-i-130supporting

confidence: 92%

Human immunodeficiency virus type 1 (HIV‐1) recombinant reverse transcriptase

Sharma¹,

Fan²,

Evans³

1994

FEBS Letters

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A recombinant p66 form of human immunodeficiency virus type 1 (HIV-l) reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl. Biochem. 14, 69-811 from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). We have analyzed the p66 HIV-l RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12. The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV-l RT (22.2 min peak). Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4°C. Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min. The protein species thus obtained were active in the RNA-dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV-l RT upon incubation with the HIV-l protease. When the IMAC-purified, imidazole-free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4"C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC. While both of these p66-containing species were stable and displayed identical RNA-dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A. These imidazole-mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.

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Section: Sk Sharma Et Al Ifebs Letters 343 (1994) 125-i-130supporting

confidence: 92%

Human immunodeficiency virus type 1 (HIV‐1) recombinant reverse transcriptase

Sharma¹,

Fan²,

Evans³

1994

FEBS Letters

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“…The survey included proteases from the following viruses: HIV-1, HIV-2, AMV (avian myeloblastosis virus), RSV (Rous sarcoma virus), HTLV-1 (human T cell leukemia virus type 1), BLV (bovine leukemia virus), Mo-MuLV (Moloney murine leukemia virus), EIAV (equine infectious anemia virus), and FIV (feline immunodeficiency virus); its outcome is summarized in Tables S1 and S2. In some cases fully denaturated proteins had been exposed to HIV-1 or HIV-2 proteases [61,62,64]. Noncleavable octapeptides were in these cases extracted from the noncleavable fragments located between the observed cleavage sites by using an eight-residue-long sliding window.…”

Section: Methodsmentioning

confidence: 99%

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

Kontijevskis¹,

Prūsis²,

Petrovska³

et al. 2005

PLoS Comput Biol

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Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular-chemical mechanisms involved in substrate cleavage by retroviral proteases.Citation: Kontijevskis A, Prusis P, Petrovska R, Yahorava S, Mutulis F, et al. (2007) A look inside HIV resistance through retroviral protease interaction maps. PLoS Comput Biol 3(3): e48.

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“…This preference was predicted to be because of hydrogen bonding of the side chain carboxyl to the backbone NH of residues 29Ј and 30Ј of the enzyme (11). Also, P 2 Ј Glu is frequently found in viral and cellular protein cleavage sites of HIV-1 proteinase (33). At the P 2 -P 2 Ј region the type 1 substrate used by Griffith et al (11) contained residues (-Asn-Tyr*ProIle-) identical to those in SP-211; however, P 2 Ј Glu substitution FIG.…”

Section: Substitutions In a Palindromic Substrate Sequencementioning

confidence: 96%

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

Tőzsér¹,

Bagossi²,

Weber³

et al. 1997

Journal of Biological Chemistry

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Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S 3 and S 3 subsites, whereas its effect is more pronounced for the S 2 and S 2 subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase.

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Human immunodeficiency virus type‐1 reverse transcriptase and ribonuclease h as substrates of the viral protease

Cited by 37 publications

References 41 publications

Human immunodeficiency virus type 1 (HIV‐1) recombinant reverse transcriptase

Human immunodeficiency virus type 1 (HIV‐1) recombinant reverse transcriptase

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

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