scholarly journals Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth

2007 ◽  
Vol 81 (21) ◽  
pp. 11798-11808 ◽  
Author(s):  
Aisling S. Dugan ◽  
Megan L. Gasparovic ◽  
Natia Tsomaia ◽  
Dale F. Mierke ◽  
Bethany A. O'Hara ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sialic Acid ◽  
Burst Size ◽  
Point Mutations ◽  
Bk Virus ◽  
Host Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Virion Protein ◽  
Virus Propagation

ABSTRACT BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing α(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.

scholarly journals Point Mutations in Herpes Simplex Virus Type 1 oriL, but Not in oriS, Reduce Pathogenesis during Acute Infection of Mice and Impair Reactivation from Latency

2006 ◽  
Vol 80 (1) ◽  
pp. 440-450 ◽  
Author(s):  
John W. Balliet ◽  
Priscilla A. Schaffer
Keyword(s):  
Herpes Simplex ◽  
Point Mutations ◽  
Tear Film ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In vitro studies of herpes simplex virus type 1 (HSV-1) viruses containing mutations in core sequences of the viral origins of DNA replication, oriL and oriS, that eliminate the ability of these origins to initiate viral-DNA synthesis have demonstrated little or no effect on viral replication in cultured cells, leading to the conclusion that the two types of origins are functionally redundant. It remains unclear, therefore, why origins that appear to be redundant are maintained evolutionarily in HSV-1 and other neurotropic alphaherpesviruses. To test the hypothesis that oriL and oriS have distinct functions in the HSV-1 life cycle in vivo, we determined the in vivo phenotypes of two mutant viruses, DoriL-ILR and DoriS-I, containing point mutations in oriL and oriS site I, respectively, that eliminate origin DNA initiation function. Following corneal inoculation of mice, tear film titers of DoriS-I were reduced relative to wild-type virus. In all other tests, however, DoriS-I behaved like wild-type virus. In contrast, titers of DoriL-ILR in tear film, trigeminal ganglia (TG), and hindbrain were reduced and mice infected with DoriL-ILR exhibited greatly reduced mortality relative to wild-type virus. In the TG explant and TG cell culture models of reactivation, DoriL-ILR reactivated with delayed kinetics and, in the latter model, with reduced efficiency relative to wild-type virus. Rescuant viruses DoriL-ILR-R and DoriS-I-R behaved like wild-type virus in all tests. These findings demonstrate that functional differences exist between oriL and oriS and reveal a prominent role for oriL in HSV-1 pathogenesis.

scholarly journals Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe

2019 ◽  
Vol 2019 ◽  
pp. 1-12
Author(s):  
Zsolt Csabai ◽  
Dóra Tombácz ◽  
Zoltán Deim ◽  
Michael Snyder ◽  
Zsolt Boldogkői
Keyword(s):  
Single Molecule ◽  
Base Composition ◽  
Pseudorabies Virus ◽  
Point Mutations ◽  
Wild Type Virus ◽  
Economic Losses ◽  
Wild Type ◽  
Southeast Europe ◽  
Long Read ◽  
National Programs

Background. Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.Results. In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.Conclusion. PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

scholarly journals Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity

1995 ◽  
Vol 311 (2) ◽  
pp. 699-704 ◽  
Author(s):  
A Muraoka ◽  
M Hashiramoto ◽  
A E Clark ◽  
L C Edwards ◽  
H Sakura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Structural Features ◽  
Transport Activity ◽  
Wild Type ◽  
C Terminus ◽  
Wild Type Clone

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.

scholarly journals NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

2015 ◽  
Vol 197 (20) ◽  
pp. 3339-3353 ◽  
Author(s):  
Jihong Li ◽  
John C. Freedman ◽  
Bruce A. McClane
Keyword(s):  
Sialic Acid ◽  
Clostridium Perfringens ◽  
Host Cells ◽  
Start Codon ◽  
Null Mutant ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Type D ◽  
Epsilon Toxin

ABSTRACTClostridium perfringenstype D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011,http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, ananInull mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due tonanIgene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions incodYandccpAgene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A doublecodYccpAnull mutant produced even less ETX than acodYorccpAsingle null mutant. CcpA bound directly to sequences upstream of theetxorcodYstart codon, and bioinformatics identified putative CcpA-bindingcresites immediately upstream of both thecodYandetxstart codons, suggesting possible direct CcpA regulatory effects. AccpAmutation also decreasedcodYtranscription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects oncodYtranscription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease.IMPORTANCEClostridium perfringensNanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using singleccpAorcodYnull mutants and accpAcodYdouble null mutant showed thatcodYandccpAregulate ETX production independently of one another but thatccpAalso affectscodYtranscription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulateetxtranscription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producingC. perfringensstrains, via CcpA and CodY, to upregulate ETX production and cause disease.

scholarly journals The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

scholarly journals Hydrophobic Amino Acids in the Human Immunodeficiency Virus Type 1 p2 and Nucleocapsid Proteins Can Contribute to the Rescue of Deleted Viral RNA Packaging Signals

2001 ◽  
Vol 75 (16) ◽  
pp. 7230-7243 ◽  
Author(s):  
Liwei Rong ◽  
Rodney S. Russell ◽  
Jing Hu ◽  
Yongjun Guan ◽  
Lawrence Kleiman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Side Chains ◽  
Wild Type ◽  
Immunodeficiency Virus

ABSTRACT An RNA fragment of 75 nucleotides, which is located between the primer binding site and the 5′ major splice donor site in human immunodeficiency virus type 1, has been shown to participate in specific encapsidation of viral RNA. Compensation studies have identified two second-site mutations, namely, MP2 (a T12I substitution in p2) and MNC (a T24I substitution in the nucleocapsid [NC] protein) that were involved in the rescue of various deletions in the aforementioned RNA region (i.e., BH-D1, BH-D2, and BH-LD3). To study whether the MP2 and MNC point mutations exert their compensatory effects in a cis manner, production of Gag proteins was blocked by insertion of stop codons into LD3, LD3-MP2-MNC, and wild-type BH10 such that the constructs generated, i.e., LD3-DG, LD3-MP2-MNC-DG, and BH-DG, only provided RNA transcripts for packaging. The results of cotransfection experiments showed that the LD3-MP2-MNC-DG viral RNA was packaged as inefficiently as LD3-DG; in contrast, BH-DG was efficiently packaged. Therefore, nucleotide substitutions in MP2 and MNC did not act in a cis manner to correct the packaging deficits in LD3. Next, we deliberately changed the T12 in p2 or the T24 in the NC to each of 19 other amino acids. We found that amino acids with long hydrophobic side chains, i.e., V, L, I, and M, were favored at either position 12 in p2 or at position 24 in NC to compensate for the above-mentioned deletions. Further studies showed that only a few amino acids could not be used at these two sites by the wild-type virus due to decreased RNA levels in the virion or abnormal Gag protein processing. In this case, W, D, and E could not substitute for T12 in p2, and S, D, and N could not substitute for T24 in NC, without affecting viral infectivity. Therefore, the long hydrophobic side chains of V, L, I, and M are necessary for these amino acids to rescue the BH-D1, BH-D2, and BH-LD3 mutated viruses.

scholarly journals Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem

2003 ◽  
Vol 77 (16) ◽  
pp. 8650-8660 ◽  
Author(s):  
Erik Burnett ◽  
Peter Tattersall
Keyword(s):  
Binding Site ◽  
Simian Virus 40 ◽  
Viral Fitness ◽  
Host Cells ◽  
Initiation Factor ◽  
Wild Type Virus ◽  
Reverse Genetic System ◽  
Wild Type ◽  
Left Hand ◽  
Factor Binding

ABSTRACT The left-hand or 3′-terminal hairpin of minute virus of mice (MVM) contains sequence elements essential for both viral DNA replication at the left-hand origin (oriL) and for the modulation of the P4 promoter, from which the viral nonstructural gene cassette is transcribed. This hairpin sequence has proven difficult to manipulate in the context of the viral genome. Here we describe a system for generating mutant viruses using synthetic hairpin oligonucleotides and a truncated form of the infectious clone. This allows manipulation of the sequence of the left-hand hairpin and examination of the effects in the context of the viral life cycle. We have confirmed the requirement for a functional parvovirus initiation factor (PIF) binding site and determined that an optimized PIF binding site, with 6 bases between the half-sites, was actually detrimental to viral growth. The distal PIF half-site overlaps a cyclic AMP-responsive element (CRE), which was shown to play an important role in initiating infection, particularly in 324K simian virus 40-transformed human fibroblasts. Interestingly, reducing the spacing of the PIF half-sites, and thus the affinity of the binding site for PIF, increased viral fitness relative to wild type in 324K cells, but not in murine A9 cells. These results indicate that the relative importance of factor binding to the CRE and PIF sites during the establishment of an infection differs markedly between these two host cells and suggest that the suboptimal spacing of PIF half-sites found in wild-type virus represents a necessary reduction in the affinity of the PIF interaction in favor of CRE function.

scholarly journals A Hypervariable Region within the 3′ cis-Acting Element of the Murine Coronavirus Genome Is Nonessential for RNA Synthesis but Affects Pathogenesis

2006 ◽  
Vol 81 (3) ◽  
pp. 1274-1287 ◽  
Author(s):  
Scott J. Goebel ◽  
Timothy B. Miller ◽  
Corey J. Bennett ◽  
Kristen A. Bernard ◽  
Paul S. Masters
Keyword(s):  
Tissue Culture ◽  
Deletion Mutant ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Hepatitis Virus ◽  
Point Mutations ◽  
Hypervariable Region ◽  
Wild Type Virus ◽  
Wild Type ◽  
Group 2

ABSTRACT The 3′ cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3′ untranslated region (3′ UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3′ UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5′-GGAAGAGC-3′, which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.

scholarly journals Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle

2006 ◽  
Vol 80 (17) ◽  
pp. 8371-8378 ◽  
Author(s):  
Xuyan Feng ◽  
Jörg Schröer ◽  
Dong Yu ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Mutant Virus ◽  
Replication Cycle ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Protein ◽  
Viral Dna ◽  
Infected Cells

ABSTRACT We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

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