scholarly journals Substitutions at Loop Regions of TMUV E Protein Domain III Differentially Impair Viral Entry and Assembly

2021 ◽  
Vol 12 ◽  
Author(s):  
Tao Hu ◽  
Zhen Wu ◽  
Shaoxiong Wu ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Virus Entry ◽  
Critical Role ◽  
Protein Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type Virus ◽  
Virus Binding ◽  
Loop Regions ◽  
Domain Iii

Flavivirus envelope protein (E) plays an important role in cellular infection, especially in virulence and antigenicity. E domain III of Tembusu virus (TMUV) is highly conserved among flaviviruses and contains four loop regions. However, the functions of the loop regions of TMUV E domain III in the viral life cycle have not yet been discovered. In this study, using a reverse genetics system, we performed site-directed mutagenesis on loops I, II, III, and IV of TMUV E domain III. Mutant 6 (S388A.G389A.K390A) showed better proliferation than the wild-type virus, while mutants 1–5 exhibited decreased in vitro infectivity, as determined by immunofluorescence assay (IFA). Based on a TMUV replicon system, the mutations exhibited no apparent effect on TMUV RNA replication. Subcellular fractionation assays and packaging system assays indicated that mutations in loops II–IV (T332A, T332S, S365A.S366A.T367A, and S388A.G389A.K390A, respectively) disrupted virion assembly. Moreover, loops I–IV played an important role in virus binding and entry, while mutant 6 (S388A.G389A.K390A) exhibited robust activity in virus entry. Taken together, our findings indicated the critical role of the loop regions in TMUV E domain III in the virus entry and assembly process.

scholarly journals Impact of Mutations at Residue I223 of the Neuraminidase Protein on the Resistance Profile, Replication Level, and Virulence of the 2009 Pandemic Influenza Virus

2011 ◽  
Vol 56 (3) ◽  
pp. 1208-1214 ◽  
Author(s):  
Andrés Pizzorno ◽  
Yacine Abed ◽  
Xavier Bouhy ◽  
Édith Beaulieu ◽  
Corey Mallett ◽  
...  
Keyword(s):  
Amino Acid ◽  
Pandemic Influenza ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Wild Type Virus ◽  
Recombinant Viruses ◽  
Viral Yield ◽  
H275y Mutation

ABSTRACTAmino acid substitutions at residue I223 of the neuraminidase (NA) protein have been identified in 2009 pandemic influenza (pH1N1) variants with altered susceptibilities to NA inhibitors (NAIs). We used reverse genetics and site-directed mutagenesis to generate the recombinant A/Québec/144147/09 pH1N1 wild-type virus (WT) and five (I223R, I223V, H275Y, I223V-H275Y, and I223R-H275Y) NA mutants. A fluorimetry-based assay was used to determine 50% inhibitory concentrations (IC50s) of oseltamivir, zanamivir, and peramivir. Replicative capacity was analyzed by viral yield assays in ST6GalI-MDCK cells. Infectivity and transmission of the WT, H275Y, and I223V-H275Y recombinant viruses were evaluated in ferrets. As expected, the H275Y mutation conferred resistance to oseltamivir (982-fold) and peramivir (661-fold) compared to the drug-susceptible recombinant WT. The single I223R mutant was associated with reduced susceptibility to oseltamivir (53-fold), zanamivir (7-fold) and peramivir (10-fold), whereas the I223V virus had reduced susceptibility to oseltamivir (6-fold) only. Interestingly, enhanced levels of resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir (1,647-, 17,347-, and 16-fold increases in IC50s, respectively) were observed for the I223R-H275Y recombinant, while the I223V-H275Y mutant exhibited 1,733-, 2,707-, and 2-fold increases in respective IC50s. The I223R and I223V changes were associated with equivalent or higher viral titersin vitrocompared to the recombinant WT. Infectivity and transmissibility in ferrets were comparable between the recombinant WT and the H275Y or I223V-H275Y recombinants. In conclusion, amino acid changes at residue I223 may alter the NAI susceptibilities of pH1N1 variants without compromising fitness. Consequently, I223R and I223V mutations, alone or with H275Y, need to be thoroughly monitored.

scholarly journals Recovery of Infectious Virus by Transfection of In Vitro-Generated RNA from Tulane Calicivirus cDNA

2008 ◽  
Vol 82 (22) ◽  
pp. 11429-11436 ◽  
Author(s):  
Chao Wei ◽  
Tibor Farkas ◽  
Karol Sestak ◽  
Xi Jiang
Keyword(s):  
Reverse Genetics ◽  
Genomic Sequence ◽  
Rhesus Macaques ◽  
Wild Type Virus ◽  
Primate Research ◽  
Complete Genomic Sequence ◽  
Subgenomic Rna ◽  
Reading Frame ◽  
Complete Genomic

ABSTRACT Tulane virus (TV) is a newly reported calicivirus that was isolated from stool samples of captive rhesus macaques from the Tulane National Primate Research Center (TNPRC). The virus has been cultivated successfully in LLC-MK2 rhesus monkey kidney cells. Its complete genomic sequence suggests that TV represents a new genus and is evolutionarily more closely related to Norovirus than to any other genus of Caliciviridae. In this study, we demonstrated that RNA transcripts made in vitro from the full-length genomic cDNA of TV were infectious upon transfection into permissive LLC-MK2 cells. The recombinant virus exhibited plaque morphologies and growth kinetics similar to those of the wild-type virus in this cell line. Capping was required for TV RNA infectivity. Although a subgenomic RNA has been detected in TV-transfected cells, a separate subgenomic RNA transcript was not required for the initial transfection to establish the replication. Transfection of truncated RNA lacking open reading frame 2 (ORF2) and ORF3 or TV-norovirus chimeric RNA resulted in abortive replication without the production of infectious progeny viruses, indicating that both ORFs are essential for the replication of TV. A heterologous insertion at the 5′ end of the genome also hampered viral replication, suggesting that an authentic 5′ end of the genome is critical for replication. The availability of the complete genomic sequence and the reverse genetics system described herein make TV a valuable model for studying calicivirus pathogenesis and replication.

scholarly journals Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs

2003 ◽  
Vol 77 (12) ◽  
pp. 7078-7092 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Subrata Barman ◽  
Tae Yong Yang ◽  
Debi P. Nayak
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Nuclear Translocation ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Plaque Morphology ◽  
Wild Type ◽  
Wild Type Phenotype

ABSTRACT Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (ΔYRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, ΔYRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.

Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid

2009 ◽  
Vol 421 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Kazuaki Ohara ◽  
Ayumu Muroya ◽  
Nobuhiro Fukushima ◽  
Kazufumi Yazaki
Keyword(s):  
Molecular Model ◽  
Substrate Binding ◽  
Expression System ◽  
Critical Role ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Hydroxybenzoic Acid ◽  
Membrane Bound

The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane α-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.

scholarly journals Duplication of the chromosomal bla SHV-11 gene in a clinical hypermutable strain of Klebsiella pneumoniae

Microbiology ◽  
2011 ◽  
Vol 157 (2) ◽  
pp. 496-503 ◽  
Author(s):  
Chloé Duvernay ◽  
Laure Coulange ◽  
Brigitte Dutilh ◽  
Véronique Dubois ◽  
Claudine Quentin ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Klebsiella Pneumoniae ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Repair System ◽  
Resistance Level ◽  
Mutator Phenotype ◽  
Single Strain

In a collection of 110 clinical isolates of Klebsiella pneumoniae, a single strain, Kp593, was found to exhibit a mutator phenotype with a rifampicin mutation frequency 100-fold higher than the modal value for this species. Complementation experiments with the wild-type MutL, one of the main components of the methyl-directed mismatch repair system, allowed the mutator phenotype to be reversed. Sequencing revealed substitution of the conserved residue Lys307 to Arg and site-directed mutagenesis followed by complementation experiments confirmed the critical role of this mutation. The patient infected with Kp593 relapsed a month later and the strain isolated then, Kp869, was identical to Kp593, as verified by PFGE analysis. Phenotypically, Kp869 colonies were more mucoid than those of Kp593, probably due to increased capsule synthesis as shown by electron microscopy. In addition, Kp869 exhibited a 16-fold higher amoxicillin resistance level related to a 36.4 kb tandem duplication encompassing the chromosomal bla SHV-11 gene, which was unstable in vitro. These data suggest that the mutator phenotype found in Kp593/Kp869 is associated with beneficial mutations conferring a selective advantage, such as increased virulence factor production and antibiotic resistance. The latter was due to resistance gene duplication, an event rarely described in natural isolates. This is the first description of the in vivo occurrence of gene duplication in a mutator background.

scholarly journals Ebola Virus VP40 Late Domains Are Not Essential for Viral Replication in Cell Culture

2005 ◽  
Vol 79 (16) ◽  
pp. 10300-10307 ◽  
Author(s):  
Gabriele Neumann ◽  
Hideki Ebihara ◽  
Ayato Takada ◽  
Takeshi Noda ◽  
Darwyn Kobasa ◽  
...  
Keyword(s):  
Cell Culture ◽  
Virus Replication ◽  
Reverse Genetics ◽  
Ebola Virus ◽  
Critical Role ◽  
Particle Formation ◽  
Late Domain ◽  
The Matrix ◽  
Late Domains

ABSTRACT Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture.

scholarly journals Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions.

1996 ◽  
Vol 16 (2) ◽  
pp. 724-734 ◽  
Author(s):  
K A Koch ◽  
D J Thiele
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Candida Glabrata ◽  
Regulatory Element ◽  
Critical Role ◽  
Minor Groove ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Binding Studies

Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity.

scholarly journals Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro

2001 ◽  
Vol 82 (7) ◽  
pp. 1785-1790 ◽  
Author(s):  
Karma Carrier ◽  
Yu Xiang ◽  
Hélène Sanfaçon
Keyword(s):  
Cleavage Site ◽  
Terminal Region ◽  
Ringspot Virus ◽  
Protein Domain ◽  
Site Directed Mutagenesis ◽  
Cdna Clones ◽  
Cleavage Sites ◽  
Tomato Ringspot Virus ◽  
Definition Of

The proteinase of Tomato ringspot virus (genus Nepovirus) is responsible for proteolytic cleavage of the RNA2-encoded polyprotein (P2) at two cleavage sites, allowing definition of the domains for the movement protein (MP) and coat protein. In this study, we have characterized a third cleavage site in the N-terminal region of P2 using an in vitro processing assay and partial cDNA clones. Results from site-directed mutagenesis of putative cleavage sites suggest that cleavage occurs at dipeptide Q301/G. Cleavage at this site is predicted to result in the release of two proteins from the N-terminal region of P2: a 34 kDa protein located at the N terminus of P2 (assuming translation initiation at the first AUG codon) and a 71 kDa protein located immediately upstream of the MP domain. In contrast, only one protein domain is present in the equivalent region of the P2 polyprotein of other characterized nepoviruses.

scholarly journals Cell Surface Proteoglycans Syndecan-1 and -4 Bind Overlapping but Distinct Sites in Laminin α3 LG45 Protein Domain

2012 ◽  
Vol 287 (15) ◽  
pp. 12204-12216 ◽  
Author(s):  
Sonia Carulli ◽  
Konrad Beck ◽  
Guila Dayan ◽  
Sophie Boulesteix ◽  
Hugues Lortat-Jacob ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Core Protein ◽  
Binding Specificity ◽  
Protein Domain ◽  
Site Directed Mutagenesis ◽  
Extracellular Matrix Protein ◽  
Heparin Binding

Keratinocyte migration during epidermal repair depends on interactions between cellular heparan sulfate proteoglycan receptors, syndecan-1 and -4, and the C-terminal globular domains (LG45) of the extracellular matrix protein laminin 332. This study investigates the molecular basis of the binding specificity of the syndecan-1 and -4 receptors expressed by human keratinocytes. We used site-directed mutagenesis to alter a recombinant LG45 protein by substituting the most critical basic residues with glutamine. All proteins were expressed in mammalian cells, purified, and characterized biochemically. We used in vitro binding assays, including surface plasmon resonance, to examine interactions between mutated LG45 and heparan sulfates, syndecan-1 and -4. We identify a major heparin binding domain on the outer edge of a β-strand of LG45 surrounded by a track of converging low affinity residues. This domain harbors distinctive syndecan-1 and -4 binding-specific sequences. This is the first study to demonstrate a binding specificity of two proteoglycans produced by a single cell type. In addition, we found that although syndecan-1 interacts exclusively through its glycosaminoglycan chains, syndecan-4 binding relies on both its core protein and its heparan sulfate chains. These results suggest that LG45 may trigger different signals toward keratinocytes depending on its interaction with syndecan-1 or -4.

scholarly journals Characterization of Dengue Virus Complex-Specific Neutralizing Epitopes on Envelope Protein Domain III of Dengue 2 Virus

2008 ◽  
Vol 82 (17) ◽  
pp. 8828-8837 ◽  
Author(s):  
Gregory D. Gromowski ◽  
Nicholas D. Barrett ◽  
Alan D. T. Barrett
Keyword(s):  
Dengue Virus ◽  
Critical Role ◽  
Antigenic Site ◽  
Protein Domain ◽  
Virus Infectivity ◽  
E Proteins ◽  
Mutant Proteins ◽  
Virus Complex ◽  
Domain Iii

ABSTRACT The surface of the mature dengue virus (DENV) particle is covered with 180 envelope (E) proteins arranged as homodimers that lie relatively flat on the virion surface. Each monomer consists of three domains (ED1, ED2, and ED3), of which ED3 contains the critical neutralization determinant(s). In this study, a large panel of DENV-2 recombinant ED3 mutant proteins was used to physically and biologically map the epitopes of five DENV complex-specific monoclonal antibodies (MAbs). All five MAbs recognized a single antigenic site that includes residues K310, I312, P332, L389, and W391. The DENV complex antigenic site was located on an upper lateral surface of ED3 that was distinct but overlapped with a previously described DENV-2 type-specific antigenic site on ED3. The DENV complex-specific MAbs required significantly higher occupancy levels of available ED3 binding sites on the virion, compared to DENV-2 type-specific MAbs, in order to neutralize virus infectivity. Additionally, there was a great deal of variability in the neutralization efficacy of the DENV complex-specific MAbs with representative strains of the four DENVs. Overall, the differences in physical binding and potency of neutralization observed between DENV complex- and type-specific MAbs in this study demonstrate the critical role of the DENV type-specific antibodies in the neutralization of virus infectivity.

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