scholarly journals Rapid Genetic Engineering of Human Cytomegalovirus by Using a Lambda Phage Linear Recombination System: Demonstration that pp28 (UL99) Is Essential for Production of Infectious Virus

2004 ◽  
Vol 78 (1) ◽  
pp. 539-543 ◽  
Author(s):  
William J. Britt ◽  
Michael Jarvis ◽  
Jun-Young Seo ◽  
Derek Drummond ◽  
Jay Nelson
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Intracellular Localization ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Base Change ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Recombination System

ABSTRACT A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.

scholarly journals Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Kalpana Dulal ◽  
Benjamin Silver ◽  
Hua Zhu
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Bac Clone ◽  
Dna Viruses ◽  
E Coli ◽  
Recombination System ◽  
Capture Method

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

scholarly journals Sequence Requirements for Localization of Human Cytomegalovirus Tegument Protein pp28 to the Virus Assembly Compartment and for Assembly of Infectious Virus

2006 ◽  
Vol 80 (11) ◽  
pp. 5611-5626 ◽  
Author(s):  
Jun-Young Seo ◽  
William J. Britt
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Tegument Protein ◽  
Reading Frame ◽  
Transfected Cells ◽  
Recombinant Viruses ◽  
Assembly Compartment ◽  
Sequence Requirements

ABSTRACT The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.

scholarly journals The Cytoplasmic Tail of Glycoprotein M (gpUL100) Expresses Trafficking Signals Required for Human Cytomegalovirus Assembly and Replication

2007 ◽  
Vol 81 (19) ◽  
pp. 10316-10328 ◽  
Author(s):  
Magdalena Krzyzaniak ◽  
Michael Mach ◽  
William J. Britt
Keyword(s):  
Human Cytomegalovirus ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Intracellular Localization ◽  
Cytoplasmic Tail ◽  
Viral Gene ◽  
Gene Encoding ◽  
Particle Assembly ◽  
Reading Frame ◽  
Virion Envelope

ABSTRACT The virion envelope of human cytomegalovirus (HCMV) is complex and consists of an incompletely defined number of glycoproteins. The gM/gN protein complex is the most abundant protein component of the envelope. Studies have indicated that deletion of the viral gene encoding either gM or gN is a lethal mutation. Analysis of the amino acid sequence of gM disclosed a C-terminal acidic cluster of amino acids and a tyrosine-containing trafficking motif, both of which are well-described trafficking/sorting signals in the cellular secretory pathway. To investigate the roles of these signals in the trafficking of the gM/gN complex during virus assembly, we made a series of gM (UL100 open reading frame) mutants in the AD169 strain of HCMV. Mutant viruses that lacked the entire C-terminal cytoplasmic tail of gM were not viable, suggesting that the cytoplasmic tail of gM is essential for virus replication. In addition, the gM mutant protein lacking the cytoplasmic domain exhibited decreased protein stability. Mutant viruses with a deletion of the acidic cluster or alanine substitutions in tyrosine-based motifs were viable but exhibited a replication-impaired phenotype suggestive of a defect in virion assembly. Analysis of these mutant gMs using static immunofluorescence and fluorescence recovery after photobleaching demonstrated delayed kinetics of intracellular localization of the gM/gN protein to the virus assembly compartment compared to the wild-type protein. These data suggest an important role of the glycoprotein gM during virus assembly, particularly in the dynamics of gM trafficking during viral-particle assembly.

scholarly journals Optimization of a Lambda-RED Recombination Method for Rapid Gene Deletion in Human Cytomegalovirus

2021 ◽  
Vol 22 (19) ◽  
pp. 10558
Author(s):  
Estéfani García-Ríos ◽  
Julia Gata-de-Benito ◽  
Mireia López-Siles ◽  
Michael J. McConnell ◽  
Pilar Pérez-Romero
Keyword(s):  
Human Cytomegalovirus ◽  
Gene Function ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Artificial Chromosome ◽  
Deletion Mutants ◽  
Transplant Patients ◽  
Recombination System ◽  
Rapid Generation ◽  
Red Recombination

Human cytomegalovirus (HCMV) continues to be a major cause of morbidity in transplant patients and newborns. However, the functions of many of the more than 282 genes encoded in the HCMV genome remain unknown. The development of bacterial artificial chromosome (BAC) technology contributes to the genetic manipulation of several organisms including HCMV. The maintenance of the HCMV BAC in E. coli cells permits the rapid generation of recombinant viral genomes that can be used to produce viral progeny in cell cultures for the study of gene function. We optimized the Lambda-Red Recombination system to construct HCMV gene deletion mutants rapidly in the complete set of tested genes. This method constitutes a useful tool that allows for the quick generation of a high number of gene deletion mutants, allowing for the analysis of the whole genome to improve our understanding of HCMV gene function. This may also facilitate the development of novel vaccines and therapeutics.

scholarly journals Role of Envelope Glycoprotein Complexes in Cell-Associated Spread of Human Cytomegalovirus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 614
Author(s):  
Nina Weiler ◽  
Caroline Paal ◽  
Kerstin Adams ◽  
Christopher Calcaterra ◽  
Dina Fischer ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Infectious Virus ◽  
Viral Envelope ◽  
Accessory Proteins ◽  
Viral Growth ◽  
Cell Association ◽  
Quantitative Immunoblotting ◽  
Model Virus

The role of viral envelope glycoproteins, particularly the accessory proteins of trimeric and pentameric gH/gL-complexes, in cell-associated spread of human cytomegalovirus (HCMV) is unclear. We aimed to investigate their contribution in the context of HCMV variants that grow in a strictly cell-associated manner. In the genome of Merlin pAL1502, the glycoproteins gB, gH, gL, gM, and gN were deleted by introducing stop codons, and the mutants were analyzed for viral growth. Merlin and recent HCMV isolates were compared by quantitative immunoblotting for expression of accessory proteins of the trimeric and pentameric gH/gL-complexes, gO and pUL128. Isolates were treated with siRNAs against gO and pUL128 and analyzed regarding focal growth and release of infectious virus. All five tested glycoproteins were essential for growth of Merlin pAL1502. Compared with this model virus, higher gO levels were measured in recent isolates of HCMV, and its knockdown decreased viral growth. Knockdown of pUL128 abrogated the strict cell-association and led to release of infectivity, which allowed cell-free transfer to epithelial cells where the virus grew again strictly cell-associated. We conclude that both trimer and pentamer contribute to cell-associated spread of recent clinical HCMV isolates and downregulation of pentamer can release infectious virus into the supernatant.

scholarly journals Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-d-Ribonucleosides

2005 ◽  
Vol 79 (23) ◽  
pp. 14660-14667 ◽  
Author(s):  
Alexandra Dittmer ◽  
John C. Drach ◽  
Leroy B. Townsend ◽  
Anke Fischer ◽  
Elke Bogner
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Unit Length ◽  
Large Subunit ◽  
Intracellular Localization ◽  
Direct Interaction ◽  
Portal Protein ◽  
Specific Association ◽  
Carboxy Terminal

ABSTRACT Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-d-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.

scholarly journals The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

2007 ◽  
Vol 81 (10) ◽  
pp. 5212-5224 ◽  
Author(s):  
Michael Mach ◽  
Karolina Osinski ◽  
Barbara Kropff ◽  
Ursula Schloetzer-Schrehardt ◽  
Magdalena Krzyzaniak ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Critical Role ◽  
Point Mutations ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Type I ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Assembly Compartment

ABSTRACT Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

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 Phenotypes of vesicular stomatitis virus mutants with mutations in the PSAP motif of the matrix protein

2012 ◽  
Vol 93 (4) ◽  
pp. 857-865 ◽  
Author(s):  
Linda Obiang ◽  
Hélène Raux ◽  
Malika Ouldali ◽  
Danielle Blondel ◽  
Yves Gaudin
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Susceptibility Gene ◽  
Point Mutations ◽  
Binding Motif ◽  
Single Mutation ◽  
Amino Terminal ◽  
Vesicular Stomatitis ◽  
Late Domains

Vesicular stomatitis virus (VSV) matrix protein (M) has a flexible amino-terminal part that recruits cellular partners. It contains a dynamin-binding site that is required for efficient virus assembly, and two motifs, 24PPPY27 and 37PSAP40, that constitute potential late domains. Late domains are present in proteins of several enveloped viruses and are involved in the ultimate step of the budding process (i.e. fission between viral and cellular membranes). In baby hamster kidney (BHK)-21 cells, it has been demonstrated that the 24PPPY27 motif binds the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) E3 ubiquitin ligase for efficient virus budding and that the 37PSAP40 motif, although conserved among M proteins of vesiculoviruses, does not possess late-domain activity. In this study, we have re-examined the contribution of the PSAP motif to VSV budding. First, we demonstrate that VSV M indeed binds TSG101 [tumour susceptibility gene 101; a component of the ESCRT1 (endosomal sorting complex required for transport 1)] through its PSAP motif. Second, we analysed the phenotype of several recombinant mutants. We show that a double mutant with point mutations in both the PSAP and the PPPY motifs is impaired compared with a single mutant in the PPPY motif, indicating that the PSAP motif partially compensates for the lack of the PPPY motif. Mutants’ phenotypes depend on cell lines: in CERA (chicken embryo-related, Alger clone) cells, a recombinant virus with a single mutation in the PSAP motif was impaired compared with the wild type, and a mutant with a single mutation in the dynamin-binding motif was much less impaired in Vero cells than in BSR (clones of BHK-21) cells. These results have implications for the VSV budding pathway that will be discussed.

Sign in / Sign up

Export Citation Format

Share Document