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

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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In VitroProcessing of HIV-1 Nucleocapsid Protein by the Viral Proteinase: Effects of Amino Acid Substitutions at the Scissile Bond in the Proximal Zinc Finger Sequence†

Biochemistry ◽

10.1021/bi035625z ◽

2004 ◽

Vol 43 (14) ◽

pp. 4304-4312 ◽

Cited By ~ 4

Author(s):

József Tözsér ◽

Sergey Shulenin ◽

John M. Louis ◽

Terry D. Copeland ◽

Stephen Oroszlan

Keyword(s):

Amino Acid ◽

Zinc Finger ◽

Nucleocapsid Protein ◽

Amino Acid Substitutions ◽

Scissile Bond ◽

Hiv 1

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Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

The Journal of Cell Biology ◽

10.1083/jcb.134.6.1455 ◽

1996 ◽

Vol 134 (6) ◽

pp. 1455-1467 ◽

Cited By ~ 116

Author(s):

B Nikolic ◽

E Mac Nulty ◽

B Mir ◽

G Wiche

Keyword(s):

Amino Acid ◽

Intermediate Filaments ◽

Basic Amino Acid ◽

Site Directed Mutagenesis ◽

Sequence Motif ◽

Amino Acid Residues ◽

Nuclear Targeting ◽

Mutant Proteins ◽

Carboxy Terminal

We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.

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Highly prevalent Russian HIV-1 V3-loop sequence variants are susceptible to maraviroc

Antiviral Chemistry and Chemotherapy ◽

10.1177/20402066211025156 ◽

2021 ◽

Vol 29 ◽

pp. 204020662110251

Author(s):

ME Lewis ◽

B Jubb ◽

P Simpson ◽

A Lopatukhin ◽

D Kireev ◽

...

Keyword(s):

Amino Acid ◽

Virologic Failure ◽

Natural Resistance ◽

V3 Loop ◽

Amino Acid Residues ◽

Subtype B ◽

Loop Sequence ◽

Hiv 1 ◽

Amino Acid Variants

Introduction Maraviroc inhibits CCR5-tropic HIV-1 across different subtypes in vitro and has demonstrated efficacy in clinical trials. V3-loop amino acid variants observed in individual maraviroc-resistant viruses have not been found to be predictive of reduced susceptibility. Sequence-database searches have demonstrated that approximately 7.3% of viruses naturally encode these variants, raising concerns regarding potential pre-existing resistance. A study from Russia reported that combinations of these same amino acids are present in the V3 loops of the Russian variant subtype A (IDU-A, now A6) with a much greater prevalence (range: 74.4%–92.3%) depending on the combination. However, these studies and database searches did not include phenotypic evaluation. Methods Sixteen Russian HIV-1 isolates (including sub-subtype A6 viruses) were assessed for V3 loop sequence and phenotypic susceptibility to maraviroc. Results All 12 of the A6 viruses and 2/4 subtype B isolates encoded V3-loop variants that have previously been identified in individual virus isolates with reduced susceptibility to maraviroc. However, despite the prevalence of these V3-loop amino acid variants among the tested viruses, phenotypic sensitivity to maraviroc was observed in all instances. Similarly, reduced susceptibility to maraviroc was not found in virus from participants who experienced virologic failure in a clinical study of maraviroc in Russia (A4001101, [NCT01275625]). Discussion Altogether, these data confirm that the presence of individual or combinations of V3-loop amino acid residues in sub-subtype A6 viruses alone does not predict natural resistance to maraviroc and that V3-loop genotype analysis of R5 virus prior to treatment is not helpful in predicting clinical outcome.

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Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in Vitro

Virology ◽

10.1016/0042-6822(92)90499-f ◽

1992 ◽

Vol 188 (2) ◽

pp. 459-468 ◽

Cited By ~ 121

Author(s):

Meera Drelich ◽

Roland Wilhelm ◽

Jan Mous

Keyword(s):

Amino Acid ◽

Amino Acid Residues ◽

Hiv 1

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Identification of a band-3 binding site near the N-terminus of erythrocyte membrane protein 4.2

Biochemical Journal ◽

10.1042/bj3090677 ◽

1995 ◽

Vol 309 (2) ◽

pp. 677-681 ◽

Cited By ~ 20

Author(s):

A C Rybicki ◽

S Musto ◽

R S Schwartz

Keyword(s):

Amino Acid ◽

Membrane Protein ◽

Binding Site ◽

Human Erythrocyte ◽

Basic Amino Acid ◽

Band 3 ◽

Amino Acid Residues ◽

Protein 4.2 ◽

Erythrocyte Plasma Membrane

Protein 4.2 (P4.2) is a major component of the erythrocyte plasma membrane accounting for approx. 5% of total membrane protein. The major membrane binding site for P4.2 is contained within the cytoplasmic domain of band 3 (cdb3), although the precise location of the cdb3 binding site is not known. To identify the cdb3 binding site, we used synthetic P4.2 peptides (15-mers) that spanned the entire 721-amino-acid large isoform of P4.2, and determined the binding of these peptides to cdb3 in an in vitro binding assay. One peptide, P8 (L61FVRRGQPFTIILYF), bound strongly to cdb3 and four others bound less strongly (P22, L271LNKRRGSVPILRQW; P27, G346EGQRGRIWIFQTST; P41, L556WRKKLHLTLSANLE; P48, I661HRERSYRFRSVWPE). These peptides have in common a cluster of two or three basic amino acid residues (arginine or lysine), in a region without nearby acidic residues. Cdb3 bound saturably to P8 with a Kd of 0.16 microM and a capacity of 0.56 mol of cdb3 monomer/mol of P8. Use of overlapping synthetic peptides further defined the cdb3 site as being contained within V63RRGQPFTIILYF. Replacement of R64R with R64G, G64R or G64G almost completely abolished cdb3 binding, suggesting that R64R is essential for cdb3 binding. P8 competitively inhibited binding of purified human erythrocyte P4.2 to cdb3. In blot overlay assays, cdb3 bound to a 23 kDa N-terminal P4.2 tryptic peptide containing V63RRGQPFTIILYF but not to other P4.2 tryptic peptides lacking this site. The V63RRGQPFTIILYF site is highly conserved in mouse and human erythrocyte P4.2 as well as between P4.2 and transglutaminase proteins, which are evolutionarily related to P4.2.

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Discovery of New Classes of Glycine transporter 2 (GlyT2) Inhibitors and Study of GlyT2 Selectivity by Combination of Novel Structural Based Virtual Screening Approach and Free Energy Perturbation (FEP+) Calculations

10.1101/510487 ◽

2019 ◽

Author(s):

Filip Fratev ◽

Manuel Miranda-Arango ◽

Elvia Padilla ◽

Suman Sirimulla

Keyword(s):

Chronic Pain ◽

Free Energy ◽

Amino Acid ◽

Virtual Screening ◽

Basic Amino Acid ◽

Free Energy Perturbation ◽

Amino Acid Residues ◽

Strong Inhibitor ◽

Energy Perturbation

In recent years, the mammalian GlyT2 transporter have emerged as a promising target for the development of anti-chronic pain agents. In our current work, we discovered a new set of promising hits that inhibit the glycine transport at nano and micromolar activity and have excellent selectivity over GlyT1 (as shown by in vitro studies), using a newly designed virtual screening (VS) protocol that combines a structure-based pharmacophore and docking screens. Furthermore, the free energy perturbation (FEP+ protocol) calculations and molecular dynamics (MD) studies revealed the GlyT2 amino acid residues critical for the binding and selectivity of both Glycine and our Lead1 compound. The FEP+ results well-matched available literature mutational data proving the quality of generated GlyT2 structure. Based on these calculations we propose that Lead1 may also be a strong inhibitor of the neutral and basic amino acid transporter B (0+) (SLC6A14). Thus, the subsequent lead optimization and characterization of refined compounds may lead to both chronic pain and pancreatic cancer agents addressing an unmet and challenging clinical needs.

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Identification of a cDNA encoding a novel amphibian growth hormone-releasing peptide and localization of its transcript

Journal of Endocrinology ◽

10.1677/joe.0.1740395 ◽

2002 ◽

Vol 174 (3) ◽

pp. 395-402 ◽

Cited By ~ 53

Author(s):

K Sawada ◽

K Ukena ◽

S Kikuyama ◽

K Tsutsui

Keyword(s):

Growth Hormone ◽

Amino Acid ◽

Cellular Localization ◽

Hormone Secretion ◽

Basic Amino Acid ◽

Pituitary Hormone ◽

Amino Acid Residues ◽

Rapid Amplification

Recently, we identified in the bullfrog brain a novel neuropeptide with a C-terminal Leu-Pro-Leu-Arg-Phe-NH(2) sequence. This amphibian neuropeptide was shown to stimulate growth hormone (GH) release in vitro and in vivo and so was designated frog GH-releasing peptide (fGRP). In this study, we cloned a cDNA encoding fGRP from the bullfrog brain by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced fGRP precursor consisted of 221 amino acid residues, encoding one fGRP and three putative fGRP-related peptides that included Leu-Pro-Xaa-Arg-Phe-NH(2) (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Northern blot analysis detected a single band of approximately 1.0 kb, indicating that no alternatively spliced forms were present. Such an apparent migration was in agreement with the estimated length of the cDNA, 902 bp. In situ hybridization further revealed the cellular localization of fGRP mRNA in the suprachiasmatic nucleus in the hypothalamus. In addition to fGRP, its related peptides may be hypothalamic factors involved in pituitary hormone secretion.

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Amino Acid Requirements of the Nucleocapsid Protein of HIV-1 for Increasing Catalytic Activity of a Ki-ras Ribozyme in Vitro

Journal of Molecular Biology ◽

10.1006/jmbi.1994.1592 ◽

1994 ◽

Vol 242 (4) ◽

pp. 422-429 ◽

Cited By ~ 30

Author(s):

Gerd Müller ◽

Bettina Strack ◽

Jens Dannull ◽

Brian S. Sproat ◽

Andrej Surovoy ◽

...

Keyword(s):

Catalytic Activity ◽

Amino Acid ◽

Nucleocapsid Protein ◽

Amino Acid Requirements ◽

Hiv 1

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Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02285-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 437-450 ◽

Cited By ~ 3

Author(s):

Takeo Kuwata ◽

Ikumi Enomoto ◽

Masanori Baba ◽

Shuzo Matsushita

Keyword(s):

Amino Acid ◽

Resistant Strain ◽

Neutralizing Antibodies ◽

High Sensitivity ◽

Ccr5 Antagonist ◽

Amino Acid Residues ◽

Resistant Variant ◽

Cd4 Binding Site ◽

Hiv 1

ABSTRACTCenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used thein vitroinduction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK652-67(strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KKWTfrom which cenicriviroc-resistant strain KK652-67was obtained was resistant to these NAbs. The V3 region of KK652-67was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK652-67resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KKWT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other.

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