Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain

ABSTRACTThe porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replicationin vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replicationin vitro, it enhanced virus replicationin vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses.IMPORTANCEThe tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replicationin vitrobut enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

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Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01432-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 436-444 ◽

Cited By ~ 1

Author(s):

Naoki Ogura ◽

Yukiyo Toyonaga ◽

Izuru Ando ◽

Kunihiro Hirahara ◽

Tsutomu Shibata ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Viral Resistance ◽

Hcv Rna ◽

Amino Acid Substitutions ◽

Sequencing Analysis ◽

Resistance Mutations ◽

In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

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TheEscherichia coliβ-Barrel Assembly Machinery Is Sensitized to Perturbations under High Membrane Fluidity

Journal of Bacteriology ◽

10.1128/jb.00517-18 ◽

2018 ◽

Vol 201 (1) ◽

Cited By ~ 5

Author(s):

Kelly M. Storek ◽

Rajesh Vij ◽

Dawei Sun ◽

Peter A. Smith ◽

James T. Koerber ◽

...

Keyword(s):

Amino Acid ◽

Membrane Proteins ◽

Outer Membrane ◽

Membrane Fluidity ◽

Amino Acid Substitutions ◽

Inhibitory Antibody ◽

Critical Determinant ◽

Gram Negative ◽

Growth Environment

ABSTRACTIntegral β-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the β-barrel assembly machine (BAM). This essential complex, composed of a β-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulatein vitro. Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of β-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σEstress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of β-barrel membrane proteins by BamA.IMPORTANCEBamA is an essential component of the β-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which β-barrel folding occurs.

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Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817455116 ◽

2018 ◽

Vol 116 (2) ◽

pp. 679-688 ◽

Cited By ~ 12

Author(s):

Ming-ling Liao ◽

George N. Somero ◽

Yun-wei Dong

Keyword(s):

Amino Acid ◽

Thermal Adaptation ◽

Site Directed Mutagenesis ◽

Dynamics Simulation ◽

Amino Acid Substitutions ◽

Thermal Stabilities ◽

Specific Amino Acid ◽

Enzyme Thermal Stability ◽

And Function

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from −1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.

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Three amino acid substitutions in the NS1 protein change the virus replication of H5N1 influenza virus in human cells

Virology ◽

10.1016/j.virol.2018.04.004 ◽

2018 ◽

Vol 519 ◽

pp. 64-73 ◽

Cited By ~ 2

Author(s):

Jing Li ◽

Kun Zhang ◽

Quanjiao Chen ◽

Xiaoshuang Zhang ◽

Yeping Sun ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Virus Replication ◽

Human Cells ◽

Amino Acid Substitutions ◽

Ns1 Protein ◽

H5n1 Influenza Virus ◽

H5n1 Influenza ◽

Protein Change

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THE METABOLISM OF ANIMAL TISSUES CULTIVATED IN VITRO: II. AMINO ACID METABOLISM OF CHICK EMBRYONIC KIDNEY, CHICK EMBRYONIC LIVER, AND MONKEY KIDNEY CORTEX CULTURES

Canadian Journal of Biochemistry and Physiology ◽

10.1139/o58-019 ◽

1958 ◽

Vol 36 (1) ◽

pp. 171-184 ◽

Cited By ~ 9

Author(s):

Arthur E. Pasieka ◽

Helen J. Morton ◽

Joseph F. Morgan

Keyword(s):

Tissue Culture ◽

Amino Acid ◽

Culture Medium ◽

Synthetic Medium ◽

Amino Acid Uptake ◽

Monkey Kidney ◽

Kidney Cortex ◽

Acid Uptake ◽

Embryonic Liver

Freshly-explanted chick embryonic kidney, chick embryonic liver, and trypsinized monkey kidney cortex cells have been cultivated in vitro in completely synthetic medium M 150. The amino acid changes in the nutrient medium during cultivation of these tissues have been studied by paper chromatography. A characteristic pattern of amino acid uptake and accumulation in the used culture medium has been demonstrated with each type of tissue culture. It has also been shown that, while the amino acid changes in the medium are different with each type of tissue culture, all cultures examined removed adenine from the medium and liberated small amounts of material thought to be hypoxanthine.

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Amino Acid Substitutions in Positions 385 and 393 of the Hydrophobic Region of VP4 May Be Associated with Rotavirus Attenuation and Cell Culture Adaptation

Viruses ◽

10.3390/v12040408 ◽

2020 ◽

Vol 12 (4) ◽

pp. 408

Author(s):

Yusheng Guo ◽

David E. Wentworth ◽

Karla M. Stucker ◽

Rebecca A. Halpin ◽

Ham Ching Lam ◽

...

Keyword(s):

Cell Culture ◽

Amino Acid ◽

Reverse Genetics ◽

Vaccine Development ◽

Nonsynonymous Substitution ◽

Amino Acid Substitutions ◽

Viral Gastroenteritis ◽

Hydrophobic Region ◽

Hydrophobic Domain ◽

Underlying Mechanisms

Rotaviruses (RVs) are the leading cause of the acute viral gastroenteritis in young children and livestock animals worldwide. Although live attenuated vaccines have been applied to control RV infection for many years, the underlying mechanisms of RV attenuation following cell culture adaption are unknown. To study these mechanisms at the genomic level, we have sequenced and conducted a comparative analysis of two virulent human (Wa, G1P[8] and M, G3P[8]) and two virulent porcine (Gottfried, G4P[6] and OSU, G5P[7]) RV strains maintained in gnotobiotic piglets for 22, 11, 12 and 9 serial passages, respectively, with their attenuated counterparts serially passaged in MA-104 cell cultures for 25, 43, 54 and 43 passages, respectively. We showed that most of the mutations were clustered in the VP4 gene, with a relatively high nonsynonymous substitution rate (81.2%). Moreover, two amino acid substitutions observed in the VP4 gene were conserved between two or more strain pairs. D385N substitution was found in M, Wa and Gottfried strains, and another one, S471H/L was present in Wa and Gottfried strains. Importantly, D385 was reported previously in another study and may be involved in regulation of virus entry. Of interest, although no 385 substitution was found in OSU strains, the attenuated OSU strain contained a unique D393H substitution within the same VP4 hydrophobic domain. Collectively, our data suggest that the VP4 hydrophobic region may play an important role in RV attenuation and aa385 and aa393 may represent potential targets for RV vaccine development using reverse genetics and site-specific mutagenesis.

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Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly of Virus-Like Particles Composed of Simian Virus 40 VP1 Capsid Protein

Journal of Virology ◽

10.1128/jvi.75.1.61-72.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 61-72 ◽

Cited By ~ 65

Author(s):

Ken-Ichiro Ishizu ◽

Hajime Watanabe ◽

Song-Iee Han ◽

Shin-Nosuke Kanesashi ◽

Mainul Hoque ◽

...

Keyword(s):

Amino Acid ◽

Calcium Ion ◽

Insect Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Ion Binding ◽

Amino Acid Substitutions ◽

Disulfide Linkage ◽

Calcium Ion Binding

ABSTRACT The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.

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Alteration of amino acids in VP2 of very virulent infectious bursal disease virus results in tissue culture adaptation and attenuation in chickens

Journal of General Virology ◽

10.1099/0022-1317-83-1-121 ◽

2002 ◽

Vol 83 (1) ◽

pp. 121-129 ◽

Cited By ~ 95

Author(s):

A. A. W. M. van Loon ◽

N. de Haas ◽

I. Zeyda ◽

E. Mundt

Keyword(s):

Amino Acids ◽

Tissue Culture ◽

Amino Acid ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Site Directed Mutagenesis ◽

Infectious Bursal Disease ◽

Single Amino Acid ◽

Culture Adaptation ◽

Bursal Disease

Reverse genetics technology offers the possibility to study the influence of particular amino acids of infectious bursal disease virus (IBDV) on adaptation to tissue culture. Genomic segments A and B of the very virulent (vv) IBDV field strain UK661 were completely cloned and sequenced, and the strain was rescued from full-length cDNA copies of both segments (UK661rev). Using site-directed mutagenesis, alteration of a single amino acid in the segment A-encoded VP2 (A284T) resulted in a limited capacity of UK661 to replicate in tissue culture. Additional alteration of a second amino acid (Q253H) increased replication efficiency in tissue culture. The second mutant (UK661-Q253H-A284T) was used to infect chickens and results were compared with UK661 and UK661rev. Whereas UK661 and UK661rev induced 100% morbidity and 50–80% mortality, UK661-Q253H-A284T proved to be strikingly attenuated, producing neither morbidity nor mortality. Moreover, UK661-Q253H-A284T-infected animals were protected from challenge infection. Thus, alteration of two specific amino acids in the VP2 region of IBDV resulted in tissue culture adaptation and attenuation in chickens of vvIBDV. The data demonstrate that VP2 plays a decisive role in pathogenicity of IBDV.

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Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication

Journal of Virology ◽

10.1128/jvi.02218-17 ◽

2018 ◽

Vol 92 (8) ◽

Cited By ~ 2

Author(s):

Jonas Kjær ◽

Graham J. Belsham

Keyword(s):

Amino Acid ◽

Disease Virus ◽

Virus Replication ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Amino Acid Substitutions ◽

2A Peptide ◽

Polyprotein Processing ◽

Mouth Disease Virus ◽

Foot And Mouth

ABSTRACTFoot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational “cleavage” of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed “ribosome skipping” or “StopGo.” Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E14, S15, and N16within the 2A sequences of infectious FMDVs despite their reported “cleavage” efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P17, G18, or P19, which displayed little or no “cleavage” activityin vitro, were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E14, S15, N16, and P19resulted in partial “cleavage” of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational “cleavage.” Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient “cleavage” at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV.IMPORTANCEFoot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational “cleavage” of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA. Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability, and polyprotein processing. They also indicate that efficient “cleavage” at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV.

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