scholarly journals The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system

2005 ◽  
Vol 86 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Detlef Michel ◽  
Irena Milotić ◽  
Markus Wagner ◽  
Bianca Vaida ◽  
Jens Holl ◽  
...  
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Ex Vivo ◽  
Stop Codon ◽  
Artificial Chromosome ◽  
Replication Cycle ◽  
Coding Region ◽  
C Terminus ◽  
Translation Stop Codon

The human cytomegalovirus (HCMV) UL78 ORF is considered to encode a seven-transmembrane receptor. However, neither the gene nor the UL78 protein has been characterized so far. The objective of this study was to investigate the UL78 gene and to clarify whether it is essential for replication. UL78 transcription was activated early after infection, was inhibited by cycloheximide but not by phosphonoacetic acid, and resulted in a 1·7 kb mRNA. Later in the replication cycle, a second mRNA of 4 kb evolved, comprising the UL77 and UL78 ORFs. The 5′ end of the UL78 mRNA initiated 48 bp upstream of the translation start and the polyadenylated tail started 268 bp downstream of the UL78 translation stop codon within the UL79 ORF. By using bacterial artificial chromosome technology, a recombinant HCMV lacking most of the UL78 coding region was constructed. Successful reconstitution of the UL78-deficient virus proved that the gene was not essential for virus replication in fibroblasts. The deletion also did not reduce virus replication in ex vivo-cultured sections of human renal arteries. Analysis of viral proteins at different stages of the replication cycle confirmed these results. Among clinical HCMV isolates, the predicted UL78 protein was highly conserved. However, an accumulation of different single mutations could be found in the N-terminal region and at the very end of the C terminus. Due to the absence of an in vivo HCMV model, the role of UL78 in the pathogenesis of HCMV infection in humans remains unclear.

scholarly journals Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

2010 ◽  
Vol 84 (18) ◽  
pp. 9019-9026 ◽  
Author(s):  
Andrea Schuessler ◽  
Kerstin Laib Sampaio ◽  
Laura Scrivano ◽  
Christian Sinzger
Keyword(s):  
Endothelial Cell ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Protein Domain ◽  
Cell Infection ◽  
Peptide Motifs ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Charge Cluster ◽  
A Charge

ABSTRACT The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.

scholarly journals Human cytomegalovirus exploits TACC3 to control microtubule dynamics and late stages of infection

2021 ◽  
Author(s):  
Colleen Furey ◽  
Helen Astar ◽  
Derek Walsh
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Coiled Coil ◽  
Microtubule Dynamics ◽  
Replication Cycle ◽  
Microtubule Organizing Center ◽  
Viral Particles ◽  
Organizing Center ◽  
Assembly Compartment ◽  
Late Stages

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less-well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. But distinct from other viruses studied to date, at mid-to-late stages of its unusually protracted replication cycle human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure termed the Assembly Compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of Transforming Acidic Coiled Coil protein 3 (TACC3) to recruit the MT polymerase, chTOG from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. Importance Diverse viruses rely on host cell microtubule networks in order to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. Yet precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the Assembly Compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid-to-late stages of infection, HCMV increases the expression of Transforming Acidic Coiled Coil protein 3 (TACC3) in order to control the localization of the microtubule polymerase, chTOG. This in turn enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.

scholarly journals Efficient Therapeutic Delivery by a Novel Cell-Penetrating Peptide Derived from Acinus

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1858
Author(s):  
Justine Habault ◽  
Claire Fraser ◽  
Ewa Pasquereau-Kotula ◽  
Maëlys Born-Bony ◽  
Anne Marie-Cardine ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Clinical Settings ◽  
Aspartic Acid Residue ◽  
Circulating Cells ◽  
C Terminus ◽  
Cell Penetrating ◽  
Endocytosis Pathway ◽  
Cancerous Cells

In this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an aspartic acid residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (residues 377–399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sézary patients’ circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sézary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings.

scholarly journals uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

2019 ◽  
Vol 47 (21) ◽  
pp. 11326-11343 ◽  
Author(s):  
Kristýna Poncová ◽  
Susan Wagner ◽  
Myrte Esmeralda Jansen ◽  
Petra Beznosková ◽  
Stanislava Gunišová ◽  
...  
Keyword(s):  
Translational Control ◽  
Stop Codon ◽  
Translation Termination ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
C Terminus ◽  
Context Specific ◽  
Stop Codon Readthrough

Abstract Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

scholarly journals Replication Properties of Clade A/C Chimeric Feline Immunodeficiency Viruses and Evaluation of Infection Kinetics in the Domestic Cat

2008 ◽  
Vol 82 (16) ◽  
pp. 7953-7963 ◽  
Author(s):  
Sohela de Rozìeres ◽  
Jesse Thompson ◽  
Magnus Sundstrom ◽  
Julia Gruber ◽  
Debora S. Stump ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Domestic Cat ◽  
Fine Tuning ◽  
Surface Glycoprotein ◽  
Wild Type ◽  
Coding Region ◽  
Mild Disease ◽  
Clade C ◽  
In The Wild

ABSTRACT Feline immunodeficiency virus (FIV) causes progressive immunodeficiency in domestic cats, with clinical course dependent on virus strain. For example, clade A FIV-PPR is predominantly neurotropic and causes a mild disease in the periphery, whereas clade C FIV-C36 causes fulminant disease with CD4+ T-cell depletion and neutropenia but no significant pathology in the central nervous system. In order to map pathogenic determinants, chimeric viruses were prepared between FIV-C36 and FIV-PPR, with reciprocal exchanges involving (i) the 3′ halves of the viruses, including the Vif, OrfA, and Env genes; (ii) the 5′ end extending from the 5′ long terminal repeat (LTR) to the beginning of the capsid (CA)-coding region; and (iii) the 3′ LTR and Rev2-coding regions. Ex vivo replication rates and in vivo replication and pathologies were then assessed and compared to those of the parental viruses. The results show that FIV-C36 replicates ex vivo and in vivo to levels approximately 20-fold greater than those of FIV-PPR. None of the chimeric FIVs recapitulated the replication rate of FIV-C36, although most replicated to levels similar to those of FIV-PPR. The rates of chloramphenicol acetyltransferase gene transcription driven by the FIV-C36 and FIV-PPR LTRs were identical. Furthermore, the ratios of surface glycoprotein (SU) to capsid protein (CA) in the released particles were essentially the same in the wild-type and chimeric FIVs. Tests were performed in vivo on the wild-type FIVs and chimeras carrying the 3′ half of FIV-C36 or the 3′ LTR and Rev2 regions of FIV-C36 on the PPR background. Both chimeras were infectious in vivo, although replication levels were lower than for the parental viruses. The chimera carrying the 3′ half of FIV-C36 demonstrated an intermediate disease course with a delayed peak viral load but ultimately resulted in significant reductions in neutrophil and CD4+ T cells, suggesting potential adaptation in vivo. Taken together, the findings suggest that the rapid-growth phenotype and pathogenicity of FIV-C36 are the result of evolutionary fine tuning throughout the viral genome, rather than being properties of any one constituent.

scholarly journals Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo

2007 ◽  
Vol 88 (11) ◽  
pp. 2941-2951 ◽  
Author(s):  
Mohammad M. Ahasan ◽  
Clive Sweet
Keyword(s):  
Virus Replication ◽  
Post Infection ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Codon Mutation

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

scholarly journals Human Cytomegalovirus Genes in the 15-Kilobase Region Are Required for Viral Replication in Implanted Human Tissues in SCID Mice

2005 ◽  
Vol 79 (4) ◽  
pp. 2115-2123 ◽  
Author(s):  
Weijia Wang ◽  
Shannon L. Taylor ◽  
Stacey A. Leisenfelder ◽  
Robert Morton ◽  
Jennifer F. Moffat ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Severe Combined Immunodeficiency ◽  
Artificial Chromosome ◽  
Scid Mice ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Human Tissues ◽  
Human Thymus ◽  
Hcmv Replication

ABSTRACT Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, ToledoBAC virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. ToledoBAC, like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (ToledoBAC) retained its virulence. The 15-kb segment was then deleted from ToledoBAC, and the resulting virus, ToledoΔ15kb, was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. ToledoΔ15kb had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the ToledoΔ15kb genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.

scholarly journals Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

2005 ◽  
Vol 280 (16) ◽  
pp. 15518-15525 ◽  
Author(s):  
Diane E. Handy ◽  
Yufeng Zhang ◽  
Joseph Loscalzo
Keyword(s):  
Glutathione Peroxidase ◽  
Untranslated Region ◽  
Stop Codon ◽  
Vascular Dysfunction ◽  
Lipid Peroxides ◽  
Mrna Levels ◽  
Coding Region ◽  
Sv40 Promoter ◽  
Secis Element

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteinein vivoand in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to “read-through” a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3′-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3′-untranslated region of the luciferase gene stimulated read-through by 6–11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p< 0.01) and decreased GPx1 enzyme activity by 45% (p= 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p< 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

334 CONSTRUCTION OF A GENETICALLY ENGINEERED PIG EXPRESSING GREEN FLUORESCENT PROTEIN (GFP)-LABELLED PROTEASOMES

2011 ◽  
Vol 23 (1) ◽  
pp. 263 ◽  
Author(s):  
C. W. O'Gorman ◽  
J. Zhao ◽  
M. S. Samuel ◽  
E. M. Walters ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Fluorescent Protein ◽  
Expressed Sequence Tag ◽  
Stop Codon ◽  
Full Length ◽  
Matrix Attachment Region ◽  
Coding Region ◽  
Public Data ◽  
Fetal Fibroblasts

Proteasomes are large protein complexes involved in protein degradation in eukaryotes and undergo dynamic redistribution between cellular compartments. Characterising the cellular localization of proteasomes at various stages of development and in response to stimuli is of interest. We hypothesised that porcine proteasomes could be visualised in vivo via a ubiquitously expressed transgene fusion comprising a proteasomal subunit and green florescent protein (GFP). The full-length sequence for porcine PSMA-1 was first constructed in silico from public data and was used to retrieve a GenBank expressed sequence tag (EST) sequence that appeared to be full length (accession CO946059; kind gift from R. S. Prather). Primers were designed to remove the stop codon and create homology for cloning with InFusion (Clontech, Palo Alto, CA, USA). The amplimer was inserted into pCAG-CreGFP (Addgene plasmid 13776) in place of the Cre coding region. The resulting plasmid (pKW14) was screened via restriction digest and sequenced for confirmation. This plasmid was confirmed functional in porcine fetal fibroblasts. After removal of the plasmid backbones, pKW14, a G418 resistance cassette (NEO), and the chicken egg white matrix attachment region were co-electroporated into male fetal fibroblasts (10 μg of total DNA, 5:2:2 ratio, respectively). Cells were grown in DMEM with 10% fetal bovine serum (FBS) and selection was initiated 36 h after transfection. Following 12 days of selection at 400 mg L–1 G418, colonies were screened by epifluorescence. Positive colonies were harvested and confirmed transgenic for all 3 input DNAs. Positive colonies were randomly pooled as sets of 3 independent integration events. Embryos were reconstructed via SCNT and transferred to 2 recipients. The fusion rates were 70 and 78%, respectively, with transfer numbers of 120 and 125 fused couplets being transferred into synchronized recipients on Day 0 of heat. Both recipients became pregnant and delivered 2 piglets each on Day 114 by Caesarean section. One live piglet was produced from each litter. Of the 2 live-born piglets, 1 survived beyond Day 3 and continues to be healthy. Transgenic status was verified by PCR. Expression was confirmed by epifluorescence of GFP-labelled proteasomes. This founder will be used to establish a model to evaluated cellular localization of proteasomes in vivo and in culture.

scholarly journals Complex Interplay of theUL136Isoforms Balances Cytomegalovirus Replication and Latency

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Katie Caviness ◽  
Farah Bughio ◽  
Lindsey B. Crawford ◽  
Daniel N. Streblow ◽  
Jay A. Nelson ◽  
...  
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Secretory Pathway ◽  
Hcmv Infection ◽  
Genetic Locus ◽  
Protein Isoforms ◽  
Hematopoietic Progenitor Cell ◽  
Transplant Patients

ABSTRACTHuman cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. TheUL136gene is carried within a genetic locus important to HCMV latency termed theUL133/8locus, which also carriesUL133,UL135, andUL138. Previously, we demonstrated thatUL136is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that theUL136isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work,UL136has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34+hematopoietic progenitor cell (HPC) model of latency, and in anin vivoNOD-scidIL2Rγcnullhumanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34+HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDaUL136isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of theUL133/8locus in HCMV infection.IMPORTANCEThe persistence of DNA viruses, and particularly of herpesviruses, remains an enigma because we have not completely defined the viral and host factors important to persistence. Human cytomegalovirus, a herpesvirus, persists in the absence of disease in immunocompetent individuals but poses a serious disease threat to transplant patients and the developing fetus. There is no vaccine, and current therapies do not target latent reservoirs. In an effort to define the viral factors important to persistence, we have studied viral genes with no known viral replication function in contexts important to HCMV persistence. Using models relevant to viral persistence, we demonstrate opposing roles of protein isoforms encoded by theUL136gene in regulating latent and replicative states of infection. Our findings reveal an intriguing interplay betweenUL136protein isoforms and defineUL136as an important regulator of HCMV persistence.

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