Glutamic Residue 438 within the Protease-Sensitive Subdomain of HIV-1 Reverse Transcriptase Is Critical for Heterodimer Processing in Viral Particles

Navarro, Jean-Marc; Damier, Laurence; Boretto, Joëlle; Priet, Stéphane; Canard, Bruno; Quérat, Gilles; Sire, Joséphine

doi:10.1006/viro.2001.1188

Cited by 9 publications

(11 citation statements)

References 16 publications

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“…Recombinant HIV-1 Molecular Clone Constructions-The 1258-bp ClaI-Eco47III fragment of the p66RTB vector containing mutations K65R, L74V, or K65R/L74V was introduced in the same restriction sites as described previously into the p66RTB-AD8 vector (20). The resulting plasmid was then digested with MscI restriction enzyme, and the 1.9-kb MscI fragment was subsequently cloned back in the same restriction sites in the pNL4.3 HIV-1 proviral molecular clone to obtain the pNL4.3 RTB plasmids mutated in the RT gene.…”

Section: Methodsmentioning

confidence: 99%

A Loss of Viral Replicative Capacity Correlates with Altered DNA Polymerization Kinetics by the Human Immunodeficiency Virus Reverse Transcriptase Bearing the K65R and L74V Dideoxynucleoside Resistance Substitutions

Deval

Navarro

Selmi

et al. 2004

Journal of Biological Chemistry

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Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2,3-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k pol : 22-fold (from 7.2 to 0.33 s ؊1 ) for K65R RT and 84-fold (from 7.2 to 0.086 s ؊1 ) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.The efficacy of current anti-HIV 1 treatments with nucleoside analogues (nucleoside reverse transcriptase inhibitors) is limited by the emergence of drug-resistant variants. Because antiretroviral therapy does not completely suppress viral replication, a resulting selection pressure favors the emergence of mutant viruses that partially or completely lose sensitivity to one or several inhibitors (1, 2). Such an example is given by the progressive appearance of primary mutations in the viral pol gene encoding the reverse transcriptase (RT) after prolonged treatments that make use of dideoxynucleosides. K65R and L74V substitutions in RT are typically associated with a moderate resistance to ddI and ddC, and these substitutions are directly responsible for a loss of efficacy of these drugs in patients carrying the corresponding mutant viruses.We previously reported the molecular role of the K65R substitution in the multiple resistance to dideoxynucleotides (3). Lysine at position 65 is located in the flexible ␤3-␤4 loop located in the finger domain of the p66 monomer of RT. The side chain of the Arg-65 interacts with the ␥-phosphate of the incoming nucleotide and alters the catalytic rate of creation of the phosphodiester bond for dideoxynucleotides specifically (3). The K65R mutation is found infrequently (less than 2%) on viral isolates from patients treated with ddI and ddC and gives a moderate 2-10-fold decrease in the sensitivity to these two drugs (4, 5). K65R also has been described to give in vitro RT-media...

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Section: Methodsmentioning

confidence: 99%

A Loss of Viral Replicative Capacity Correlates with Altered DNA Polymerization Kinetics by the Human Immunodeficiency Virus Reverse Transcriptase Bearing the K65R and L74V Dideoxynucleoside Resistance Substitutions

Deval

Navarro

Selmi

et al. 2004

Journal of Biological Chemistry

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“…Processing at this site has been estimated to occur in about 50% of the GagPol molecules and produces a truncated, 51-kDa form of the 66-kDa full-length RT that is missing the carboxy-terminal RNase H domain (12,26,30,47,50,52).…”

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confidence: 99%

Ordered Processing of the Human Immunodeficiency Virus Type 1 GagPol Precursor Is Influenced by the Context of the Embedded Viral Protease

Pettit¹,

Clemente

Jeung

et al. 2005

J Virol

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Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.

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“…Like the HIV-1 p51 protein, the HTLV-1 p49 protein cannot catalyze reverse transcription by itself; RT activity requires the catalytic p66 subunit. Pol processing is controlled in part by Pol structure, since there are mutations in HIV-1 Pol that cause overprocessing and result in the accumulation of an excess of p51 (46,47). Regardless of whether HTLV-1 is a monomer or heterodimer, the overprocessing of HTLV-1 Pol to yield predominantly p49 and low amounts of p62 RT could underlie the relatively low polymerase activity and poor infectivity of cell-free virus particles.…”

Section: Discussionmentioning

confidence: 99%

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

Mitchell

Tőzsér

Princler

et al. 2006

Journal of Biological Chemistry

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It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).Human T-cell lymphotropic virus type-1 (HTLV-1) 2 is an oncogenic retrovirus belonging to the deltaretrovirus genus, which includes HTLV-2, -3, and -4, simian T-cell lymphotropic viruses, and bovine leukemia virus. As with many other retroviruses, HTLV-1 uses ribosomal frameshifting to regulate the relative expression of Gag proteins and the viral replication enzymes. In many retroviruses, including human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV), protein synthesis that initiates from the gag start codon gives rise to both Gag and Gag-Pol polyprotein precursors. A ribosomal frameshift site encoded either at the 5Ј-end of pol (HIV) or at the 3Ј-end of gag (RSV) allows the synthesis of the Gag-Pol polyprotein (1, 2). In contrast, the pro gene of some retroviruses, such as HTLV, mouse mammary tumor virus, and Mason-Pfizer monkey virus, is in a separate reading frame from both gag and pol. Thus, the expression of Gag-Pol polyprotein in these retroviruses involves two ribosomal frameshifts: one where gag and pro overlap and the second within the pro/pol overlap (3-11). In these viruses, three polyprotein precursors are synthesized. In HTLV-1, they are Gag (Pr53), Gag-Pro (Pr76), and Gag-Pro-Pol (Pr180). Based on in vitro translation of viral RNA, the molar ratio of the Gag, Gag-Pro, and Gag-Pro-Pol polyproteins was estimated to be about 100:10:1 (12). This means that the relative amounts of Pol to Gag produced by HTLV-1 would be 5-10-fold less than the amount present in singleframeshift retroviruses, such as HIV and RSV (1, 2) and 3-4-fold less than the amount found in the other two-frameshift retroviruses: mouse mammary tumor virus and Mason-Pfizer monkey virus (13-15).Retroviral pol genes encode RT and integrase (IN), which are required for the replication and integration of the viral genome. The Pol precursor is enzymatically cleaved by the viral protease to yield the subunits of the RT complex. The reverse transcriptases of HIV-1, RSV, and murine leukemia virus are t...

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Glutamic Residue 438 within the Protease-Sensitive Subdomain of HIV-1 Reverse Transcriptase Is Critical for Heterodimer Processing in Viral Particles

Cited by 9 publications

References 16 publications

A Loss of Viral Replicative Capacity Correlates with Altered DNA Polymerization Kinetics by the Human Immunodeficiency Virus Reverse Transcriptase Bearing the K65R and L74V Dideoxynucleoside Resistance Substitutions

A Loss of Viral Replicative Capacity Correlates with Altered DNA Polymerization Kinetics by the Human Immunodeficiency Virus Reverse Transcriptase Bearing the K65R and L74V Dideoxynucleoside Resistance Substitutions

Ordered Processing of the Human Immunodeficiency Virus Type 1 GagPol Precursor Is Influenced by the Context of the Embedded Viral Protease

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

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