In vitro folding of phage P22 coat protein with amino acid substitutions that confer in vivo temperature sensitivity

Biochemistry ◽  
1995 ◽  
Vol 34 (20) ◽  
pp. 6815-6826 ◽  
Author(s):  
Carolyn M. Teschke ◽  
Jonathan King
Keyword(s):  
Amino Acid ◽  
Coat Protein ◽  
Temperature Sensitivity ◽  
Amino Acid Substitutions ◽  
Phage P22

scholarly journals Use of a Two-Color Genetic Screen To Identify a Domain of the Global Regulator Lrp That Is Specifically Required forpap Phase Variation

1998 ◽  
Vol 180 (5) ◽  
pp. 1224-1231 ◽  
Author(s):  
Linda Kaltenbach ◽  
Bruce Braaten ◽  
Julie Tucker ◽  
Margareta Krabbe ◽  
David Low
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Phase Variation ◽  
Genetic Screen ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Global Regulator ◽  
Transcription In Vitro

ABSTRACT The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as ilvIH, activation ofpapBA transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate ilvIH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on ilvIH transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for pap and ilvIH DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp’s were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity forpap Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-lockedpap derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp’s are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on pap transcription state.

scholarly journals Specific Substitutions in Region V2 of gp120 env confer SHIV Neutralisation Resistance

Pathogens ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 181
Author(s):  
Yalcin Pisil ◽  
Zafer Yazici ◽  
Hisatoshi Shida ◽  
Shuzo Matsushita ◽  
Tomoyuki Miura
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitutions ◽  
In Vitro Mutagenesis ◽  
Tier 2 ◽  
Infected Monkey ◽  
Tier 1 ◽  
V3 Region ◽  
Monkey Plasma

A tier 2 SHIV-MK38 strain was obtained after two in vivo passages of tier 1 SHIV-MK1. SHIV-MK38#818, cloned from the MK38 strain, was neutralisation-resistant, like the parental MK38 strain, to SHIV-infected monkey plasma (MP), HIV-1-infected human pooled plasma (HPP), and KD247 monoclonal antibody (mAb) (anti-V3 gp120 env). We investigated the mechanisms underlying the resistance of #818, specifically the amino acid substitutions that confer resistance to MK1. We introduced amino acid substitutions in the MK1 envelope by in vitro mutagenesis and then compared the neutralisation resistance to MP, HPP, and KD247 mAb with #818 in a neutralisation assay using TZM-bl cells. We selected 11 substitutions in the V1, V2, C2, V4, C4, and V5 regions based on the alignment of env of MK1 and #818. The neutralisation resistance of the mutant MK1s with 7 of 11 substitutions in the V1, C2, C4, and V5 regions did not change significantly. These substitutions did not alter any negative charges or N-glycans. The substitutions N169D and K187E, which added negative charges, and S190N in the V2 region of gp120 and A389T in V4, which created sites for N-glycan, conferred high neutralisation resistance. The combinations N169D+K187E, N169D+S190N, and N169D+A389T resulted in MK1 neutralisation resistance close to that of #818. The combinations without 169D were neutralisation-sensitive. Therefore, N169D is the most important substitution for neutralisation resistance. This study demonstrated that although the V3 region sequences of #818 and MK1 are the same, V3 binding antibodies cannot neutralise #818 pseudovirus. Instead, mutations in the V2 and V4 regions inhibit the neutralisation of anti-V3 antibodies. We hypothesised that 169D and 190N altered the MK1 Env conformation so that the V3 region is buried. Therefore, the V2 region may block KD247 from binding to the tip of the V3 region.

scholarly journals Virus Susceptibility Analyses from a Phase IV Clinical Trial of Inhaled Zanamivir Treatment in Children Infected with Influenza

2013 ◽  
Vol 57 (4) ◽  
pp. 1677-1684 ◽  
Author(s):  
Phillip J. Yates ◽  
Nalini Mehta ◽  
Joseph Horton ◽  
Margaret Tisdale
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Drug Pressure ◽  
Receptor Binding Site ◽  
Virus Susceptibility ◽  
Susceptibility Data

ABSTRACTA zanamivir postapproval efficacy study was conducted in children (n= 279) in Japan during three influenza seasons. Pharyngeal swab specimens (n= 714) were obtained for detailed resistance analysis. From 371 cultured viruses, 3 viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at day 1 (before treatment), 1 had an N74S amino acid substitution (fold shift, 46), and 2 (day 1 and day 2) had a Q136K amino acid substitution (fold shifts, 292 and 301). Q136K was detected only in cultured virus and not in the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. Neuraminidase (NA) population sequencing showed that viruses from 12 subjects had emergent amino acid substitutions, but 3 with susceptibility data were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. Hemagglutinin (HA) sequencing showed that viruses from 20 subjects had 9 HA amino acid substitutions that were previously implicated in resistance to neuraminidase inhibitors inin vitroassays or that were close to the receptor binding site. Their role inin vivoresistance appears to be less important but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones from 90 subjects were analyzed. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or are just chance mutations introduced during PCR.

Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis

2019 ◽  
Vol 74 (8) ◽  
pp. 2230-2238 ◽  
Author(s):  
Florent Morio ◽  
Lisa Lombardi ◽  
Ulrike Binder ◽  
Cédric Loge ◽  
Estelle Robert ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Clinical Isolate ◽  
Antifungal Susceptibility ◽  
Azole Resistance ◽  
Amino Acid Substitutions ◽  
Fluconazole Resistance ◽  
Candida Orthopsilosis

AbstractBackgroundAzoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis.ObjectivesWe investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis.MethodsAntifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate.ResultsCompared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole.ConclusionsOur data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.

scholarly journals Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

2012 ◽  
Vol 86 (16) ◽  
pp. 8602-8613 ◽  
Author(s):  
Tomokazu Tamura ◽  
Yoshihiro Sakoda ◽  
Fumi Yoshino ◽  
Takushi Nomura ◽  
Naoki Yamamoto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Amino Acid Substitutions ◽  
Virulence Determinants ◽  
Live Attenuated Vaccine

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE−was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE−vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE−/P-11 virus isolated from the tonsils after the 11th passagein vivohad acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studiesin vitroindicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiencyin vitroand pathogenicity in pigs.

scholarly journals Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

2006 ◽  
Vol 17 (10) ◽  
pp. 4282-4299 ◽  
Author(s):  
Wenyu Wen ◽  
Lu Chen ◽  
Hao Wu ◽  
Xin Sun ◽  
Mingjie Zhang ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Amino Acid ◽  
Magnetic Resonance ◽  
Adaptor Protein ◽  
Resonance Spectroscopy ◽  
Amino Acid Substitutions ◽  
Sorting Signals ◽  
Distinct Role

Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXXΦ-like adaptin-dependent sorting signal (Y31GTI34) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y31GTI34 (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y31GTI34 motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXXΦ-like sorting signals.

scholarly journals The CaaX Proteases, Afc1p and Rce1p, Have Overlapping but Distinct Substrate Specificities

2000 ◽  
Vol 20 (12) ◽  
pp. 4381-4392 ◽  
Author(s):  
Cynthia Evans Trueblood ◽  
Victor L. Boyartchuk ◽  
Elizabeth A. Picologlou ◽  
David Rozema ◽  
C. Dale Poulter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Single Amino Acid ◽  
In Vitro Assays ◽  
Sequence Motif ◽  
Amino Acid Substitutions ◽  
In The Wild

ABSTRACT Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a1, the a2, or the X position of the a-factor Ca1a2X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a1 position, V, L, I, C, or M at the a2 position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a1 substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

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