Development of a gene capture method to rescue a large deletion mutant of human cytomegalovirus

2009 ◽  
Vol 157 (2) ◽  
pp. 180-187 ◽  
Author(s):  
Kalpana Dulal ◽  
Zhen Zhang ◽  
Hua Zhu
Keyword(s):  
Human Cytomegalovirus ◽  
Deletion Mutant ◽  
Large Deletion ◽  
Gene Capture ◽  
Capture Method

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 The Carboxyl-Terminal Region of Human Cytomegalovirus IE1491aa Contains an Acidic Domain That Plays a Regulatory Role and a Chromatin-Tethering Domain That Is Dispensable during Viral Replication

2005 ◽  
Vol 79 (1) ◽  
pp. 225-233 ◽  
Author(s):  
Jens Reinhardt ◽  
Geoffrey B. Smith ◽  
Christopher T. Himmelheber ◽  
Jane Azizkhan-Clifford ◽  
Edward S. Mocarski
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Deletion Mutant ◽  
Viral Gene Expression ◽  
Mutant Virus ◽  
Serine Phosphorylation ◽  
Viral Gene ◽  
Acidic Domain ◽  
Carboxyl Terminal

ABSTRACT The human cytomegalovirus major immediate-early (α) protein IE1491aa plays an important role in controlling viral gene expression at low multiplicities of infection. With a transient complementation assay, full-length IE1491aa enhanced the growth of ie1 mutant virus CR208 20-fold better than a deletion mutant lacking 71 carboxyl-terminal amino acids (IE11-420aa). A 16-amino-acid domain between amino acids 476 and 491 was both necessary and sufficient for chromatin-tethering activity; however, this domain was completely dispensable for complementation of CR208 replication. The proximal 55-amino-acid acidic domain (amino acids 421 to 475) was found to be most important for function. A deletion mutant lacking only this domain retained chromatin-tethering activity but failed to complement mutant virus. Interestingly, serine phosphorylation (at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455) was not required for complementation. These results show that IE1491aa is composed of at least two domains that support replication, a region located between amino acids 1 and 399 that complements ie1 mutant virus replication to low levels and an acidic domain between amino acids 421 and 479 that dramatically enhances complementation.

scholarly journals Deletion of Open Reading Frame UL26 from the Human Cytomegalovirus Genome Results in Reduced Viral Growth, Which Involves Impaired Stability of Viral Particles

2006 ◽  
Vol 80 (11) ◽  
pp. 5423-5434 ◽  
Author(s):  
Kerstin Lorz ◽  
Heike Hofmann ◽  
Anja Berndt ◽  
Nina Tavalai ◽  
Regina Mueller ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Artificial Chromosome ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Transcriptional Activation Domain ◽  
Viral Particles

ABSTRACT We previously showed that open reading frame (ORF) UL26 of human cytomegalovirus, a member of the US22 multigene family of betaherpesviruses, encodes a novel tegument protein, which is imported into cells in the course of viral infection. Moreover, we demonstrated that pUL26 contains a strong transcriptional activation domain and is capable of stimulating the major immediate-early (IE) enhancer-promoter. Since this suggested an important function of pUL26 during the initiation of the viral replicative cycle, we sought to ascertain the relevance of pUL26 by construction of a viral deletion mutant lacking the UL26 ORF using the bacterial artificial chromosome mutagenesis procedure. The resulting deletion virus was verified by PCR, enzyme restriction, and Southern blot analyses. After infection of human foreskin fibroblasts, the UL26 deletion mutant showed a small-plaque phenotype and replicated to significantly lower titers than wild-type or revertant virus. In particular, we noticed a striking decrease of infectious titers 7 days postinfection in a multistep growth experiment, whereas the release of viral DNA from infected cells was not impaired. A further investigation of this aspect revealed a significantly diminished stability of viral particles derived from the UL26 deletion mutant. Consistent with this, we observed that the tegument composition of the deletion mutant deviates from that of the wild-type virus. We therefore hypothesize that pUL26 plays a role not only in the onset of IE gene transcription but also in the assembly of the viral tegument layer in a stable and correct manner.

scholarly journals A Recombinant Human Cytomegalovirus with a Large Deletion in UL97 Has a Severe Replication Deficiency

1999 ◽  
Vol 73 (7) ◽  
pp. 5663-5670 ◽  
Author(s):  
Mark N. Prichard ◽  
Ning Gao ◽  
Sanju Jairath ◽  
Gilbert Mulamba ◽  
Paula Krosky ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Large Deletion ◽  
Primary Cells ◽  
Parent Virus ◽  
Recombinant Viruses ◽  
Large Deletions ◽  
Good Target ◽  
Primary Fibroblasts ◽  
Antiviral Chemotherapy

ABSTRACT Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitivity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RCΔ97, that contain large deletions in this gene and carry a 4.8-kb insertion containing a selectable genetic marker. These mutant viruses were isolated by using a population of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impaired in their ability to replicate in primary fibroblasts, attaining virus titers that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruses retained the ability to form small, slowly growing plaques in primary fibroblasts, demonstrating that UL97 is not absolutely essential for replication in cell culture. The replication deficit was relieved when UL97 was provided in trans in the complementing cell line, showing that the phenotype was due to a deficiency in UL97. Thus, theUL97 gene product plays a very important role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.

Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation

1984 ◽  
Vol 4 (8) ◽  
pp. 1661-1663
Author(s):  
L Sompayrac ◽  
K J Danna
Keyword(s):  
Dna Sequences ◽  
Deletion Mutant ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Large Deletion ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Coding Sequence ◽  
Mouse Cells

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.

scholarly journals UL26-Deficient Human Cytomegalovirus Produces Virions with Hypophosphorylated pp28 Tegument Protein That Is Unstable within Newly Infected Cells

2006 ◽  
Vol 80 (7) ◽  
pp. 3541-3548 ◽  
Author(s):  
Joshua Munger ◽  
Dong Yu ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Deletion Mutant ◽  
Open Reading Frame ◽  
Early Gene ◽  
Immediate Early ◽  
Tegument Protein ◽  
Protein Accumulation ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Infected Cells

ABSTRACT The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.

scholarly journals Creation of a Large Deletion Mutant of Campylobacter jejuni Reveals that the Lipooligosaccharide Gene Cluster Is Not Required for Viability

2009 ◽  
Vol 191 (7) ◽  
pp. 2392-2399 ◽  
Author(s):  
Gemma L. Marsden ◽  
Jianjun Li ◽  
Paul H. Everest ◽  
Andrew J. Lawson ◽  
Julian M. Ketley
Keyword(s):  
Gene Cluster ◽  
Campylobacter Jejuni ◽  
Deletion Mutant ◽  
Natural Transformation ◽  
Large Deletion ◽  
Host Cells ◽  
The Core ◽  
Increased Sensitivity

ABSTRACT Deletion of the lipooligosaccharide biosynthesis region (Cj1132c to Cj1152c) from the genome of Campylobacter jejuni NCTC11168 shows that the core is not required for viability. The mutant was attenuated for growth and has increased sensitivity to antibiotics and detergents. Natural transformation and invasion of cultured host cells was abolished.

scholarly journals Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation.

1984 ◽  
Vol 4 (8) ◽  
pp. 1661-1663 ◽  
Author(s):  
L Sompayrac ◽  
K J Danna
Keyword(s):  
Dna Sequences ◽  
Deletion Mutant ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Large Deletion ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Coding Sequence ◽  
Mouse Cells

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.

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