scholarly journals Experimental estimation of the effects of all amino-acid mutations to HIV Env

2016 ◽  
Author(s):  
Hugh K. Haddox ◽  
Adam S. Dingens ◽  
Jesse D. Bloom
Keyword(s):  
Cell Culture ◽  
Amino Acid ◽  
Neutralizing Antibodies ◽  
Purifying Selection ◽  
Sequence Evolution ◽  
Broadly Neutralizing Antibodies ◽  
Naturally Occurring ◽  
Rapid Sequence ◽  
Amino Acid Mutations

AbstractHIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV’s most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV’s growth in cell culture. We compare our experimental measurements of each site’s preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. We show that some regions of Env have a high inherent tolerance to mutation, whereas other regions (such as epitopes of broadly neutralizing antibodies) have a significantly reduced capacity to tolerate mutations. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env’s evolution.

scholarly journals Accurate measurement of the effects of all amino-acid mutations to influenza hemagglutinin

2016 ◽  
Author(s):  
Michael B. Doud ◽  
Jesse D. Bloom
Keyword(s):  
Amino Acid ◽  
Neutralizing Antibodies ◽  
Viral Evolution ◽  
Point Mutations ◽  
Helper Virus ◽  
Broadly Neutralizing Antibodies ◽  
Amino Acid Mutation ◽  
Influenza Hemagglutinin ◽  
Amino Acid Mutations ◽  
Evolutionary Paths

AbstractInfluenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We previously used high-throughput mutagenesis and deep sequencing to estimate the effects of all mutations to an H1 influenza hemagglutinin on viral replication in cell culture (Thyagarajan and Bloom, 2014); however, these measurements suffered from sub-stantial noise. Here we describe advances that greatly improve the accuracy and reproducibility of our measurements. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. However, other regions – including stalk epitopes targeted by broadly neutralizing antibodies – have a limited capacity to evolve. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.

scholarly journals The role of amino acid α38 in the control of oxygen binding to human adult and embryonic haemoglobin Portland

1999 ◽  
Vol 343 (3) ◽  
pp. 681-685 ◽  
Author(s):  
Tao ZHENG ◽  
Thomas BRITTAIN ◽  
Nicholas J. WATMOUGH ◽  
Roy E. WEBER
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Significant Contribution ◽  
Molecular Model ◽  
Site Directed Mutagenesis ◽  
Oxygen Binding ◽  
Naturally Occurring ◽  
Two State Model ◽  
T State

The role of the amino acid at position α38 in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position α38 (which is totally conserved in all mammals) is changed to a Gln, the equilibrium properties of the protein are significantly altered. Equilibrium and kinetic data show that the R-state properties of the protein are essentially unaffected by the mutation whilst the allosteric equilibrium and T-state properties are changed. Mutation of the naturally occurring Gln38 of the human embryonic haemoglobin ζ-chain (the only known non-Thr containing globin) to a Thr residue shows the converse change in properties produced by the adult mutation, although in this case the situation is complicated by significant chain heterogeneity in the T state. An extension of the two-state model of co-operativity is presented to describe quantitatively the equilibrium ligand binding in the presence of T-state chain heterogeneity. A molecular model is described in which the putative interaction of αGln38 and βTyr145 is identified which make a significant contribution to the previously reported unusual ligand-binding properties of the ζ-chain containing human embryonic haemoglobins.

scholarly journals A VH1-69 antibody lineage from an infected Chinese donor potently neutralizes HIV-1 by targeting the V3 glycan supersite

2020 ◽  
Vol 6 (38) ◽  
pp. eabb1328 ◽  
Author(s):  
Sonu Kumar ◽  
Bin Ju ◽  
Benjamin Shapero ◽  
Xiaohe Lin ◽  
Li Ren ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Neutralizing Antibodies ◽  
Critical Role ◽  
Viral Escape ◽  
Germline Gene ◽  
Broadly Neutralizing Antibodies ◽  
Functional Studies ◽  
Single Genome ◽  
Hiv 1

An oligomannose patch around the V3 base of HIV-1 envelope glycoprotein (Env) is recognized by multiple classes of broadly neutralizing antibodies (bNAbs). Here, we investigated the bNAb response to the V3 glycan supersite in an HIV-1–infected Chinese donor by Env-specific single B cell sorting, structural and functional studies, and longitudinal analysis of antibody and virus repertoires. Monoclonal antibodies 438-B11 and 438-D5 were isolated that potently neutralize HIV-1 with moderate breadth, are encoded by the VH1-69 germline gene, and have a disulfide-linked long HCDR3 loop. Crystal structures of Env-bound and unbound antibodies revealed heavy chain–mediated recognition of the glycan supersite with a unique angle of approach and a critical role of the intra-HCDR3 disulfide. The mechanism of viral escape was examined via single-genome amplification/sequencing and glycan mutations around the N332 supersite. Our findings further emphasize the V3 glycan supersite as a prominent target for Env-based vaccine design.

scholarly journals Neutralizing Antibodies Targeting HIV-1 gp41

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1210
Author(s):  
Christophe Caillat ◽  
Delphine Guilligay ◽  
Guidenn Sulbaran ◽  
Winfried Weissenhorn
Keyword(s):  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Somatic Mutations ◽  
Heptad Repeat ◽  
Broadly Neutralizing Antibodies ◽  
Vaccine Research ◽  
Membrane Components ◽  
And Function ◽  
Hiv 1

HIV-1 vaccine research has obtained an enormous boost since the discovery of many broadly neutralizing antibodies (bnAbs) targeting all accessible sites on the HIV-1 envelope glycoprotein (Env). This in turn facilitated high-resolution structures of the Env glycoprotein in complex with bnAbs. Here we focus on gp41, its highly conserved heptad repeat region 1 (HR1), the fusion peptide (FP) and the membrane-proximal external region (MPER). Notably, the broadest neutralizing antibodies target MPER. Both gp41 HR1 and MPER are only fully accessible once receptor-induced conformational changes have taken place, although some studies suggest access to MPER in the close to native Env conformation. We summarize the data on the structure and function of neutralizing antibodies targeting gp41 HR1, FP and MPER and we review their access to Env and their complex formation with gp41 HR1, MPER peptides and FP within native Env. We further discuss MPER bnAb binding to lipids and the role of somatic mutations in recognizing a bipartite epitope composed of the conserved MPER sequence and membrane components. The problematic of gp41 HR1 access and MPER bnAb auto- and polyreactivity is developed in the light of inducing such antibodies by vaccination.

scholarly journals Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization

2016 ◽  
Vol 113 (16) ◽  
pp. 4446-4451 ◽  
Author(s):  
Jordan R. Willis ◽  
Jessica A. Finn ◽  
Bryan Briney ◽  
Gopal Sapparapu ◽  
Vidisha Singh ◽  
...  
Keyword(s):  
Amino Acid ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Computational Design ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Amino Acid Length ◽  
Massive Scale ◽  
Amino Acid Mutations ◽  
Hiv 1

Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1–naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1–naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 107unique HCDR3 amino acid regions from 70 different HIV-1–naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1–naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts.

scholarly journals Role of HIV membrane in neutralization by two broadly neutralizing antibodies

2009 ◽  
Vol 106 (48) ◽  
pp. 20234-20239 ◽  
Author(s):  
S. M. Alam ◽  
M. Morelli ◽  
S. M. Dennison ◽  
H.-X. Liao ◽  
R. Zhang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Broadly Neutralizing Antibodies

scholarly journals Correlation of In Vitro Susceptibilities to Newer Quinolones of Naturally Occurring Quinolone-Resistant Neisseria gonorrhoeae Strains with Changes in GyrA and ParC

2001 ◽  
Vol 45 (3) ◽  
pp. 734-738 ◽  
Author(s):  
Tiffany R. Shultz ◽  
John W. Tapsall ◽  
Peter A. White
Keyword(s):  
Amino Acid ◽  
Neisseria Gonorrhoeae ◽  
Southeast Asia ◽  
Point Mutations ◽  
The Other ◽  
Naturally Occurring ◽  
Ciprofloxacin Resistance ◽  
Amino Acid Mutations ◽  
Sydney Australia

ABSTRACT The in vitro activities of ciprofloxacin, trovafloxacin, moxifloxacin, and grepafloxacin against 174 strains of Neisseria gonorrhoeae isolated in Sydney, Australia, were determined. The strains included 84 quinolone-less-sensitive and -resistant N. gonorrhoeae (QRNG) strains for which ciprofloxacin MICs were in the range of 0.12 to 16 μg/ml. The QRNG included strains isolated from patients whose infections were acquired in a number of countries, mostly in Southeast Asia. The gyrA and parCquinolone resistance-determining regions (QRDR) of 18 selected QRNG strains were sequenced, and the amino acid mutations observed were related to the MICs obtained. The activities of moxifloxacin and grepafloxacin against QRNG were comparable to that of ciprofloxacin. Trovafloxacin was more active than the other quinolones against some but not all of the QRNG strains. Increments in ciprofloxacin resistance occurred in a step-wise manner with point mutations initiated ingyrA resulting in amino acid alterations Ser91-to-Phe, Ser91-to-Tyr, Asp95-to-Gly, and Asp95-to-Asn. Single gyrAchanges correlated with ciprofloxacin MICs in the range 0.12 to 1 μg/ml. The Ser91 changes in GyrA were associated with higher MICs and further QRDR changes. QRNG strains for which ciprofloxacin MICs were greater than 1 μg/ml had both gyrA and parCQRDR point mutations. ParC alterations were seen in these isolates only in the presence of GyrA changes and comprised amino acid changes Asp86-to-Asn, Ser87-to-Asn, Ser87-to-Arg, Ser88-to-Pro, Glu91-to-Lys, and Glu91-to-Gln. QRNG strains for which MICs were in the higher ranges had double GyrA mutations, but again only with accompanying ParC alterations. Not only did the nature and combination of GyrA and ParC changes influence the incremental increases in ciprofloxacin MICs, but they seemingly also altered the differential activity of trovafloxacin. Our findings suggest that the newer quinolones of the type examined are unlikely to be useful replacements for ciprofloxacin in the treatment of gonorrhea, particularly where ciprofloxacin MICs are high or where resistance is widespread.

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