scholarly journals Genetic content of wild-type human cytomegalovirus

2004 ◽  
Vol 85 (5) ◽  
pp. 1301-1312 ◽  
Author(s):  
Aidan Dolan ◽  
Charles Cunningham ◽  
Ralph D. Hector ◽  
Aycan F. Hassan-Walker ◽  
Lydia Lee ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Nucleotide Substitution ◽  
Cell Tropism ◽  
Wild Type ◽  
Single Nucleotide ◽  
Protein Coding ◽  
Glycoprotein Gene ◽  
Low Passage ◽  
Strain Ad169 ◽  
Coding Potential

The genetic content of wild-type human cytomegalovirus was investigated by sequencing the 235 645 bp genome of a low passage strain (Merlin). Substantial regions of the genome (genes RL1–UL11, UL105–UL112 and UL120–UL150) were also sequenced in several other strains, including two that had not been passaged in cell culture. Comparative analyses, which employed the published genome sequence of a high passage strain (AD169), indicated that Merlin accurately reflects the wild-type complement of 165 genes, containing no obvious mutations other than a single nucleotide substitution that truncates gene UL128. A sizeable subset of genes exhibits unusually high variation between strains, and comprises many, but not all, of those that encode proteins known or predicted to be secreted or membrane-associated. In contrast to unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in one or both of two groups of genes that may influence cell tropism. One comprises UL128, UL130 and UL131A, which putatively encode secreted proteins, and the other contains RL5A, RL13 and UL9, which are members of the RL11 glycoprotein gene family. The case in support of a lack of protein-coding potential in the region between UL105 and UL111A was also strengthened.

scholarly journals Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

2009 ◽  
Vol 84 (5) ◽  
pp. 2597-2609 ◽  
Author(s):  
Brent J. Ryckman ◽  
Marie C. Chase ◽  
David C. Johnson
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Null Mutant ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Biochemical Studies ◽  
Infected Cells ◽  
Low Passage ◽  
Er Export ◽  
Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.

scholarly journals Codon Substitution Mutations at Two Positions in the L Polymerase Protein of Human Parainfluenza Virus Type 1 Yield Viruses with a Spectrum of Attenuation In Vivo and Increased Phenotypic Stability In Vitro

2004 ◽  
Vol 78 (4) ◽  
pp. 2029-2036 ◽  
Author(s):  
Josephine M. McAuliffe ◽  
Sonja R. Surman ◽  
Jason T. Newman ◽  
Jeffrey M. Riggs ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Substitution ◽  
Wild Type ◽  
Phenotypic Stability ◽  
Single Nucleotide ◽  
Codon Substitution ◽  
Substitution Mutations ◽  
Human Parainfluenza Virus

ABSTRACT The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.

scholarly journals The Human Cytomegalovirus UL116 Glycoprotein Is a Chaperone to Control gH-Based Complexes Levels on Virions

2021 ◽  
Vol 12 ◽  
Author(s):  
Giacomo Vezzani ◽  
Diego Amendola ◽  
Dong Yu ◽  
Sumana Chandramouli ◽  
Elisabetta Frigimelica ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Wild Type Virus ◽  
Cell Tropism ◽  
Wild Type ◽  
Viral Membrane ◽  
Viral Glycoproteins ◽  
Fusion Glycoprotein ◽  
Infected Cells ◽  
Viral Membrane Fusion

Human cytomegalovirus (HCMV) relies in large part upon the viral membrane fusion glycoprotein B and two alternative gH/gL complexes, gH/gL/gO (Trimer) and gH/gL/UL128/UL130/UL131A (Pentamer) to enter into cells. The relative amounts of Trimer and Pentamer vary among HCMV strains and contribute to differences in cell tropism. Although the viral ER resident protein UL148 has been shown to interact with gH to facilitate gO incorporation, the mechanisms that favor the assembly and maturation of one complex over another remain poorly understood. HCMV virions also contain an alternative non-disulfide bound heterodimer comprised of gH and UL116 whose function remains unknown. Here, we show that disruption of HCMV gene UL116 causes infectivity defects of ∼10-fold relative to wild-type virus and leads to reduced expression of both gH/gL complexes in virions. Furthermore, gH that is not covalently bound to other viral glycoproteins, which are readily detected in wild-type HCMV virions, become undetectable in the absence of UL116 suggesting that the gH/UL116 complex is abundant in virions. We find evidence that UL116 and UL148 interact during infection indicating that the two proteins might cooperate to regulate the abundance of HCMV gH complexes. Altogether, these results are consistent with a role of UL116 as a chaperone for gH during the assembly and maturation of gH complexes in infected cells.

scholarly journals Construction of a Self-Excisable Bacterial Artificial Chromosome Containing the Human Cytomegalovirus Genome and Mutagenesis of the Diploid TRL/IRL13 Gene

2002 ◽  
Vol 76 (5) ◽  
pp. 2316-2328 ◽  
Author(s):  
Dong Yu ◽  
Gregory A. Smith ◽  
Lynn W. Enquist ◽  
Thomas Shenk
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Wild Type ◽  
Coding Region ◽  
Reading Frame ◽  
Strain Ad169

ABSTRACT The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.

scholarly journals The human cytomegalovirus UL116 glycoprotein is a chaperone to control gH-based complexes levels on virions

2020 ◽  
Author(s):  
Giacomo Vezzani ◽  
Diego Amendola ◽  
Dong Yu ◽  
Sumana Chandramuli ◽  
Elisabetta Frigimelica ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Wild Type Virus ◽  
Cell Tropism ◽  
Wild Type ◽  
Viral Membrane ◽  
Viral Glycoproteins ◽  
Fusion Glycoprotein ◽  
Infected Cells ◽  
Viral Membrane Fusion

ABSTRACTHuman cytomegalovirus (HCMV) relies in large part upon the viral membrane fusion glycoprotein B (gB) and two alternative gH/gL complexes, gH/gL/gO (Trimer) and the gH/gL/UL128/UL130/UL131A (Pentamer) to enter into cells. The relative amounts of the Trimer and Pentamer vary among HCMV strains and contribute to differences in cell tropism. Although the viral ER resident protein UL148 has been shown to interact with gH to facilitate gO incorporation, the mechanisms that favor the assembly and maturation of one complex over another remain poorly understood. HCMV virions also contain an alternative non-disulfide bound heterodimer comprised of gH and UL116 whose function remains unknown. Here, we show that disruption of HCMV gene UL116 causes infectivity defects of ~10-fold relative to wild-type virus and leads to reduced expression of both gH/gL complexes in virions. Furthermore, gH that is not covalently bound to other viral glycoproteins, which are readily detected in wild-type HCMV virions, become undetectable in the absence of UL116 suggesting that the gH/UL116 complex is abundant in virions. We find evidence that UL116 and UL148 interact during infection indicating that the two proteins might cooperate to regulate the abundance of HCMV gH complexes. Altogether, these results are consistent with a role of UL116 as a chaperone for gH during the assembly and maturation of gH complexes in infected cells.

scholarly journals Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection

2001 ◽  
Vol 75 (20) ◽  
pp. 9623-9632 ◽  
Author(s):  
Irmgard Pult ◽  
Nathan Abbott ◽  
Yong-Yuan Zhang ◽  
Jesse Summers
Keyword(s):  
Nucleotide Substitution ◽  
Acute Liver Injury ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Single Nucleotide ◽  
Reading Frame ◽  
Duck Hepatitis ◽  
B Virus ◽  
Infected Cells

ABSTRACT In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 × 10−5 to 6 × 10−5 per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 × 10−5 to 4.5 × 10−5per nucleotide per generation.

scholarly journals High Frequency of a Single Nucleotide Substitution (c.-6-180T>G) of the CanineMDR1/ABCB1Gene Associated with Phenobarbital-Resistant Idiopathic Epilepsy in Border Collie Dogs

2013 ◽  
Vol 35 ◽  
pp. 669-672 ◽  
Author(s):  
Keijiro Mizukami ◽  
Akira Yabuki ◽  
Hye-Sook Chang ◽  
Mohammad Mejbah Uddin ◽  
Mohammad Mahbubur Rahman ◽  
...  
Keyword(s):  
High Frequency ◽  
Nucleotide Substitution ◽  
Fragment Length Polymorphism ◽  
Idiopathic Epilepsy ◽  
Single Nucleotide Substitution ◽  
Wild Type ◽  
Single Nucleotide ◽  
High Mutation Frequency ◽  
High Prevalence ◽  
Mutant Homozygote

A single nucleotide substitution (c.-6-180T>G) associated with resistance to phenobarbital therapy has been found in the canineMDR1/ABCB1gene in Border Collies with idiopathic epilepsy. In the present study, a PCR-restriction fragment length polymorphism assay was developed for genotyping this mutation, and a genotyping survey was carried out in a population of 472 Border Collies in Japan to determine the current allele frequency. The survey demonstrated the frequencies of the T/T wild type, T/G heterozygote, and G/G mutant homozygote to be 60.0%, 30.3%, and 9.8%, respectively, indicating that the frequency of the mutant G allele is extremely high (24.9%) in Border Collies. The results suggest that this high mutation frequency of the mutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies.

scholarly journals Deletion of gpUL132, a Structural Component of Human Cytomegalovirus, Results in Impaired Virus Replication in Fibroblasts

2005 ◽  
Vol 79 (18) ◽  
pp. 11837-11847 ◽  
Author(s):  
Simone Spaderna ◽  
Barbara Kropff ◽  
Yvonne Ködel ◽  
Siyuan Shen ◽  
Scott Coley ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Protein Product ◽  
Viral Glycoprotein ◽  
Transcription Analysis ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Theoretical Mass ◽  
Infected Cells ◽  
Low Passage ◽  
Strain Ad169

ABSTRACT The coding capacity of human cytomegalovirus (HCMV) for glycoproteins by far exceeds that of other herpesviruses. Few of these proteins have been characterized so far. We have investigated the gene product of reading frame UL132. The putative protein product of UL132 is a glycoprotein with a theoretical mass of 29.8 kDa. Transcription analysis revealed that the gene is transcribed with a true late kinetics from the laboratory-adapted strain AD169 and the low-passage isolate TB40E. Two proteins of 22 to 28 kDa and 45 to 60 kDa were detected in virus-infected cells as well as in extracellular virions. The larger protein carried N-linked carbohydrates. Both protein forms were present in laboratory-adapted strains as well as in low-passage isolates of HCMV. Recombinant viruses with the UL132 gene deleted were constructed in the low-passage HCMV isolate PAN as well as the high-passage isolate AD169. Deletion of UL132 from either genome resulted in a pronounced replication deficit with a reduction of approximately 100-fold for HCMV strain AD169. Thus, the protein product of the UL132 reading frame represents a structural viral glycoprotein of HCMV that has an important function for viral replication in tissue culture.

scholarly journals Role of human cytomegalovirus UL131A in cell type-specific virus entry and release

2006 ◽  
Vol 87 (9) ◽  
pp. 2451-2460 ◽  
Author(s):  
Barbara Adler ◽  
Laura Scrivano ◽  
Zsolt Ruzcics ◽  
Brigitte Rupp ◽  
Christian Sinzger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Human Cytomegalovirus ◽  
Virus Entry ◽  
Cell Tropism ◽  
Virus Release ◽  
Wild Type ◽  
Cell Infection ◽  
Infected Cells ◽  
In Cis

The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169 with wild-type UL131A in cis in an ectopic position restored endothelial cell tropism. The UL131A protein was found in virions in a complex with gH. Coinfection of fibroblasts with UL131A-negative and -positive viruses restored the endothelial cell tropism of UL131A-negative virions by complementing the virions with UL131A protein. Virus entry into endothelial cells, but not into fibroblasts, was blocked by an antipeptide antiserum to pUL131A. AD169, cis-complemented with wild-type UL131A, showed an impaired release of infectious particles from fibroblasts. A comparable defect in virus release was observed when UL131A was expressed ectopically in a virus background already expressing an intact copy of UL131A. In contrast, virus release from infected endothelial cells was not affected by UL131A. These data suggest a dual role for pUL131A in virus entry and virus exit from infected cells.

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