scholarly journals Crystal Structure of a Retroviral Polyprotein: Prototype Foamy Virus Protease-Reverse Transcriptase (PR-RT)

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1495
Author(s):  
Jerry Joe E. K. Harrison ◽  
Steve Tuske ◽  
Kalyan Das ◽  
Francesc X. Ruiz ◽  
Joseph D. Bauman ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reverse Transcriptase ◽  
Foamy Virus ◽  
Polymerase Activity ◽  
Rnase H ◽  
Proteolytic Processing ◽  
Nucleic Acid Binding ◽  
Foamy Viruses ◽  
Polyprotein Precursor ◽  
Hiv 1

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.

scholarly journals Crystal structure of prototype foamy virus (PFV) protease-reverse transcriptase fusion (PR-RT) reveals conformational plasticity: implications for function

2020 ◽  
Author(s):  
Jerry Joe E. K. Harrison ◽  
Steve Tuske ◽  
Kalyan Das ◽  
Francesc X. Ruiz ◽  
Joseph D. Bauman ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reverse Transcriptase ◽  
Foamy Virus ◽  
Rnase H ◽  
Proteolytic Processing ◽  
Nucleic Acid Binding ◽  
Foamy Viruses ◽  
Conformational Plasticity ◽  
Polyprotein Precursor ◽  
Hiv 1

AbstractProteolytic processing of the retroviral Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN), except in foamy viruses (FVs) where only the IN domain is released. Here, we report the 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) needed for processing and reverse transcription. The monomeric PFV PR exhibits similar architecture as the HIV-1 PR but the N- and C-terminal residues are unstructured. A C-terminal extension of the PR folds into two helices that supports the RT palm subdomain and anchors the PR next to the RT. The subdomains of RT: fingers, palm, thumb, and connection, and the RNase H domain, are connected by flexible linkers and spatially arranged similarly to those in the HIV-1 RT p51 subunit. Significant spatial and conformational domain rearrangements are required for nucleic acid binding. This offers structural insight into retroviral RT conformational maturation and architecture of immature enzymes.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

Disruption of a Salt Bridge between Asp 488 and Lys 465 in HIV-1 Reverse Transcriptase Alters Its Proteolytic Processing and Polymerase Activity

Virology ◽  
1993 ◽  
Vol 196 (2) ◽  
pp. 731-738 ◽  
Author(s):  
Laura Goobar-Larsson ◽  
Kristina Bäckbro ◽  
Torsten Unge ◽  
Ramagauri Bhikhabhai ◽  
Lotta Vrang ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Salt Bridge ◽  
Polymerase Activity ◽  
Proteolytic Processing ◽  
Hiv 1

scholarly journals Efavirenz enhances the proteolytic processing of an HIV-1 pol polyprotein precursor and reverse transcriptase homodimer formation

FEBS Letters ◽  
2004 ◽  
Vol 579 (2) ◽  
pp. 379-384 ◽  
Author(s):  
Gilda Tachedjian ◽  
Katie L. Moore ◽  
Stephen P. Goff ◽  
Nicolas Sluis-Cremer
Keyword(s):  
Reverse Transcriptase ◽  
Proteolytic Processing ◽  
Polyprotein Precursor ◽  
Hiv 1

scholarly journals HIV-1 Reverse Transcriptase Still Remains a New Drug Target: Structure, Function, Classical Inhibitors, and New Inhibitors with Innovative Mechanisms of Actions

2012 ◽  
Vol 2012 ◽  
pp. 1-23 ◽  
Author(s):  
Francesca Esposito ◽  
Angela Corona ◽  
Enzo Tramontano
Keyword(s):  
Reverse Transcriptase ◽  
Drug Target ◽  
Polymerase Activity ◽  
Rnase H ◽  
Primary Target ◽  
Lethal Mutagenesis ◽  
Rt Inhibitors ◽  
Process Characteristic ◽  
Hiv 1 ◽  
Chain Terminators

During the retrotranscription process, characteristic of all retroviruses, the viral ssRNA genome is converted into integration-competent dsDNA. This process is accomplished by the virus-coded reverse transcriptase (RT) protein, which is a primary target in the current treatments for HIV-1 infection. In particular, in the approved therapeutic regimens two classes of drugs target RT, namely, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). Both classes inhibit the RT-associated polymerase activity: the NRTIs compete with the natural dNTP substrate and act as chain terminators, while the NNRTIs bind to an allosteric pocket and inhibit polymerization noncompetitively. In addition to these two classes, other RT inhibitors (RTIs) that target RT by distinct mechanisms have been identified and are currently under development. These include translocation-defective RTIs, delayed chain terminators RTIs, lethal mutagenesis RTIs, dinucleotide tetraphosphates, nucleotide-competing RTIs, pyrophosphate analogs, RT-associated RNase H function inhibitors, and dual activities inhibitors. This paper describes the HIV-1 RT function and molecular structure, illustrates the currently approved RTIs, and focuses on the mechanisms of action of the newer classes of RTIs.

scholarly journals Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Paul L. Boyer ◽  
Steven J. Smith ◽  
Xue Zhi Zhao ◽  
Kalyan Das ◽  
Kevin Gruber ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Nucleoside Analogs ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv Replication ◽  
Content Type ◽  
Rnase Hi ◽  
Hiv 1

ABSTRACT We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds. IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

scholarly journals Characterization of the Polymerase and RNase H Activities of Human Foamy Virus Reverse Transcriptase

2004 ◽  
Vol 78 (12) ◽  
pp. 6112-6121 ◽  
Author(s):  
Paul L. Boyer ◽  
Carolyn R. Stenbak ◽  
Patrick K. Clark ◽  
Maxine L. Linial ◽  
Stephen H. Hughes
Keyword(s):  
Reverse Transcriptase ◽  
Leukemia Virus ◽  
Foamy Virus ◽  
Polymerase Activity ◽  
Rnase H ◽  
Immunodeficiency Virus ◽  
Moloney Leukemia Virus ◽  
Basic Loop

ABSTRACT Foamy virus (FV) replication, while related to that of orthoretroviruses, differs at a number of steps. Several of these differences involve the reverse transcriptase (RT). There appear to be fewer RTs present in FV than in orthoretroviruses; we previously proposed that the polymerase of FV RT was more active than orthoretroviral RTs to compensate for the numerical difference. Here we present further characterization of the RT of FV. The polymerase activity of FV RT was greater than that of human immunodeficiency virus type 1 RT in a variety of assays. We also examined the RNase H activity of FV RT, and we propose that FV RT has a basic loop in the RNase H domain. Although the sequence of the basic loop of FV RT is different from the basic loop of either Moloney leukemia virus RNase H or Escherichia coli RNase H, the FV RT basic loop appears to have a similar function.

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