Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

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Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

The EMBO Journal ◽

10.1002/j.1460-2075.1994.tb06414.x ◽

1994 ◽

Vol 13 (7) ◽

pp. 1525-1533 ◽

Cited By ~ 127

Author(s):

J. Dannull ◽

A. Surovoy ◽

G. Jung ◽

K. Moelling

Keyword(s):

Amino Acid ◽

Zinc Finger ◽

Nucleocapsid Protein ◽

Specific Binding ◽

Basic Amino Acid ◽

Amino Acid Residues ◽

Hiv 1 ◽

Psi Rna

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Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

Journal of Virology ◽

10.1128/jvi.00229-06 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5413-5422 ◽

Cited By ~ 36

Author(s):

Aleksei Lulla ◽

Valeria Lulla ◽

Kairit Tints ◽

Tero Ahola ◽

Andres Merits

Keyword(s):

Life Cycle ◽

Amino Acid ◽

Cleavage Site ◽

Semliki Forest Virus ◽

Nonstructural Protein ◽

Cleavage Sites ◽

Vitro Assay ◽

Virus Life Cycle ◽

Cleavage Efficiency

ABSTRACT The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1′ had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1′, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.

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Cloning and functional characterization of the smooth muscle ether-a-go-go-related gene K+ channel. Potential role of a conserved amino acid substitution in the S4 region. Vol. 278 (2003) 2503-2514

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)67993-5 ◽

2004 ◽

Vol 279 (46) ◽

pp. 48486

Author(s):

Fouzia Shoeb ◽

Anna P. Malykhina ◽

Hamid I. Akbarali

Keyword(s):

Smooth Muscle ◽

Amino Acid ◽

Amino Acid Substitution ◽

Potential Role ◽

Functional Characterization ◽

K Channel ◽

Related Gene ◽

Channel Potential

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Potential role of tyrosine kinase inhibitors during primary HIV-1 infection

Expert Review of Anti-infective Therapy ◽

10.1080/14787210.2017.1308823 ◽

2017 ◽

Vol 15 (5) ◽

pp. 421-423 ◽

Cited By ~ 2

Author(s):

Juan Ambrosioni ◽

Mayte Coiras ◽

José Alcamí ◽

José M. Miró

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Potential Role ◽

Kinase Inhibitors ◽

Hiv 1

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H5N2 Avian Influenza Outbreak in Texas in 2004: the First Highly Pathogenic Strain in the United States in 20 Years?

Journal of Virology ◽

10.1128/jvi.79.17.11412-11421.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 11412-11421 ◽

Cited By ~ 67

Author(s):

Chang-Won Lee ◽

David E. Swayne ◽

Jose A. Linares ◽

Dennis A. Senne ◽

David L. Suarez

Keyword(s):

United States ◽

Amino Acid ◽

Avian Influenza ◽

Cleavage Site ◽

Basic Amino Acid ◽

The United States ◽

Influenza Viruses ◽

Single Point Mutation ◽

Highly Pathogenic

ABSTRACT In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.

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Phenotypic Susceptibility to Bevirimat in Isolates from HIV-1-Infected Patients without Prior Exposure to Bevirimat

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01784-09 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2345-2353 ◽

Cited By ~ 39

Author(s):

Nicolas A. Margot ◽

Craig S. Gibbs ◽

Michael D. Miller

Keyword(s):

Recombinant Viruses ◽

Gag Polyprotein ◽

New Class ◽

Major Mutation ◽

Hiv Drugs ◽

The Impact ◽

First Time ◽

Hiv 1

ABSTRACT Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

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Role of cysteine in jaundice hemorrhages

Kazan medical journal ◽

10.17816/kazmj52766 ◽

1935 ◽

Vol 31 (8-9) ◽

pp. 1114-1114

Keyword(s):

Amino Acid ◽

Obstructive Jaundice ◽

Blood Clotting ◽

In Vitro Experiments ◽

Main Factor

The role of calcium, platelets, and other factors that were associated with bleeding was not confirmed in jaundice. Carr and Toote found that the amino acid cysteine was the main factor impeding blood clotting. In obstructive jaundice, this amino acid accumulates in the blood and in in vitro experiments and, when administered to animals, causes a deterioration in blood clotting.

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The balance between p53 isoforms modulates the efficiency of HIV-1 infection in macrophages

Journal of Virology ◽

10.1128/jvi.01188-21 ◽

2021 ◽

Author(s):

Yann Breton ◽

Corinne Barat ◽

Michel J. Tremblay

Keyword(s):

Virus Replication ◽

Potential Role ◽

Host Factors ◽

Fine Tuning ◽

Mrna Levels ◽

Cellular Environment ◽

P53 Isoforms ◽

Antiviral Role ◽

Hiv 1

Several host factors influence HIV-1 infection and replication. The p53-mediated antiviral role in monocytes-derived macrophages (MDMs) was previously highlighted. Indeed, an increase in p53 level results in a stronger restriction against HIV-1 early replication steps through SAMHD1 activity. In this study, we investigated the potential role of some p53 isoforms in HIV-1 infection. Transfection of isoform-specific siRNA induces distinctive effects on the virus life cycle. For example, in contrast to a siRNA targeting all isoforms, a knockdown of Δ133p53 transcripts reduces virus replication in MDMs that is correlated with a decrease in phosphorylated inactive SAMHD1. Combination of Δ133p53 knockdown and Nutlin-3, a pharmacological inhibitor of MDM2 that stabilizes p53, further reduces susceptibility of MDMs to HIV-1 infection, thus suggesting an inhibitory role of Δ133p53 towards p53 antiviral activity. In contrast, p53β knockdown in MDMs increases the viral production independently of SAMHD1. Moreover, experiments with a Nef-deficient virus show that this viral protein plays a protective role against the antiviral environment mediated by p53. Finally, HIV-1 infection affects the expression pattern of p53 isoforms by increasing p53β and p53γ mRNA levels while stabilizing the protein level of p53α and some isoforms from the p53β subclass. The balance between the various p53 isoforms is therefore an important factor in the overall susceptibility of macrophages to HIV-1 infection, fine-tuning the p53 response against HIV-1. This study brings a new understanding of the complex role of p53 in virus replication processes in myeloid cells. Importance As of today, HIV-1 is still considered as a global pandemic without a functional cure, partly because of the presence of stable viral reservoirs. Macrophages constitute one of these cell reservoirs, contributing to the viral persistence. Studies investigating the host factors involved in cell susceptibility to HIV-1 infection might lead to a better understanding of the reservoir formation and will eventually allow the development of an efficient cure. Our team previously showed the antiviral role of p53 in macrophages, which acts by compromising the early steps of HIV-1 replication. In this study, we demonstrate the involvement of p53 isoforms, which regulates p53 activity and define the cellular environment influencing viral replication. In addition, the results concerning the potential role of p53 in antiviral innate immunity could be transposed to other fields of virology and suggest that knowledge in oncology can be applied to HIV-1 research.

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Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial adhesivity of human red blood cells

Thrombosis and Haemostasis ◽

10.1160/th16-05-0373 ◽

2017 ◽

Vol 117 (07) ◽

pp. 1402-1411 ◽

Cited By ~ 5

Author(s):

Laura Beth Mann Dosier ◽

Vikram J. Premkumar ◽

Hongmei Zhu ◽

Izzet Akosman ◽

Michael F. Wempe ◽

...

Keyword(s):

Amino Acid ◽

Red Blood Cells ◽

Blood Cells ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Neutral Amino Acid Transporter ◽

System L

SummaryThe system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

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