An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity.

ABSTRACT Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

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Novel Gag-Pol Frameshift Site in Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

Journal of Virology ◽

10.1128/jvi.72.7.6146-6150.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 6146-6150 ◽

Cited By ~ 52

Author(s):

Louise Doyon ◽

Catherine Payant ◽

Léa Brakier-Gingras ◽

Daniel Lamarre

Keyword(s):

Human Immunodeficiency Virus ◽

Protease Inhibitors ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cleavage Site ◽

Messenger Rna ◽

Site Mutation ◽

Ribosomal Frameshifting ◽

Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring −1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.

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Proteolysis of an active site peptide of lactate dehydrogenase by human immunodeficiency virus type 1 protease

Biochemistry ◽

10.1021/bi00157a003 ◽

1992 ◽

Vol 31 (42) ◽

pp. 10153-10168 ◽

Cited By ~ 29

Author(s):

Thaddeus A. Tomaszek ◽

Michael L. Moore ◽

James E. Strickler ◽

Robert L. Sanchez ◽

J. Scott Dixon ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Lactate Dehydrogenase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Active Site ◽

Immunodeficiency Virus

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Initial Cleavage of the Human Immunodeficiency Virus Type 1 GagPol Precursor by Its Activated Protease Occurs by an Intramolecular Mechanism

Journal of Virology ◽

10.1128/jvi.78.16.8477-8485.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8477-8485 ◽

Cited By ~ 83

Author(s):

Steven C. Pettit ◽

Lorraine E. Everitt ◽

Sumana Choudhury ◽

Ben M. Dunn ◽

Andrew H. Kaplan

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Active Site ◽

Enzyme Activation ◽

Particle Assembly ◽

Precursor Processing ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.

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Extended nucleocapsid protein is cleaved from the Gag–Pol precursor of human immunodeficiency virus type 1

Journal of General Virology ◽

10.1099/0022-1317-82-3-581 ◽

2001 ◽

Vol 82 (3) ◽

pp. 581-590 ◽

Cited By ~ 10

Author(s):

Nissim Chen ◽

Abraham Morag ◽

Nava Almog ◽

Immanuel Blumenzweig ◽

Orna Dreazin ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cleavage Site ◽

Amino Acid Residues ◽

Site Mutation ◽

Reading Frame ◽

C Terminus ◽

Immunodeficiency Virus

Human immunodeficiency virus type 1 Gag and Gag–Pol precursors are translated from an mRNA which is indistinguishable from the full-length genomic RNA. The ratio of Gag to Gag–Pol polyproteins is approximately 20:1 and is controlled by a frameshift of the reading frame, which takes place downstream of the p7 nucleocapsid (NC) in the N terminus of the p1 peptide. The viral precursors Gag and Gag–Pol are cleaved by the virus-encoded protease (PR) into the structural proteins, and into p6Pol, PR, reverse transcriptase and integrase. Due to the frameshift event, the cleavage site at the C terminus of NC coded in the Gag frame (ERQAN-FLGKI) changes either to ERQANFLRED or ERQANFFRED. The results presented in this report demonstrate that the NC released from the Gag–Pol precursor is 8 amino acid residues longer than the NC cleaved from the Gag polyprotein. Our results also show that truncated Gag–Pol precursors bearing cleavage site mutation at the NC/p6Pol, and/or p6Pol/PR junctions, undergo autoprocessing in bacterial and eukaryotic cells, indicating that PR is active when part of the precursor.

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Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00056-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 4086-4093 ◽

Cited By ~ 17

Author(s):

Angela Corona ◽

Anna Schneider ◽

Kristian Schweimer ◽

Paul Rösch ◽

Birgitta M. Wöhrl ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Active Site ◽

Foamy Virus ◽

Rnase H ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTRNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg2+needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data,in silicodocking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H.

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