scholarly journals Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA

1999 ◽  
Vol 73 (7) ◽  
pp. 5473-5480 ◽  
Author(s):  
Yuntao Wu ◽  
Ge Liu ◽  
Eric B. Carstens
Keyword(s):  
Dna Replication ◽  
Plasmid Dna ◽  
Viral Genome ◽  
Serial Passage ◽  
Viral Dna ◽  
Common Procedure ◽  
Rolling Circle ◽  
Large Deletions ◽  
Putative Origin ◽  
Integration Sites

ABSTRACT Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.

scholarly journals Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

2012 ◽  
Vol 86 (18) ◽  
pp. 9817-9827 ◽  
Author(s):  
Alexandra Nitzsche ◽  
Charlotte Steinhäußer ◽  
Katrin Mücke ◽  
Christina Paulus ◽  
Michael Nevels
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Histone Modification ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Histone H3 ◽  
Viral Dna ◽  
The Impact ◽  
Preferential Incorporation

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using “click chemistry” revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.

scholarly journals Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

2010 ◽  
Vol 84 (12) ◽  
pp. 6153-6162 ◽  
Author(s):  
Mei Yu ◽  
Eric B. Carstens
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Viral Genome ◽  
Virus Production ◽  
Autographa Californica ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Budded Virus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.

scholarly journals Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

2009 ◽  
Vol 83 (13) ◽  
pp. 6641-6651 ◽  
Author(s):  
Ayumi Kudoh ◽  
Satoko Iwahori ◽  
Yoshitaka Sato ◽  
Sanae Nakayama ◽  
Hiroki Isomura ◽  
...  
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
Viral Genome ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Recombinational Repair ◽  
Viral Dna ◽  
Barr Virus ◽  
Homologous Recombinational Repair ◽  
Epstein Barr

ABSTRACT Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

scholarly journals Bromodomain Protein 4 Mediates the Papillomavirus E2 Transcriptional Activation Function

2006 ◽  
Vol 80 (9) ◽  
pp. 4276-4285 ◽  
Author(s):  
Michal-Ruth Schweiger ◽  
Jianxin You ◽  
Peter M. Howley
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Viral Genome ◽  
Regulatory Protein ◽  
Activation Function ◽  
Transactivation Domain ◽  
Genome Maintenance ◽  
Mitotic Chromosomes ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen: Replicating and Shielding Viral DNA during Viral Persistence

2017 ◽  
Vol 91 (14) ◽  
Author(s):  
Magdalena Weidner-Glunde ◽  
Giuseppe Mariggiò ◽  
Thomas F. Schulz
Keyword(s):  
Dna Replication ◽  
Viral Genome ◽  
Kaposi's Sarcoma ◽  
Viral Latency ◽  
Viral Persistence ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Current View ◽  
Dna Binding Domain ◽  
Viral Dna

ABSTRACT Kaposi's sarcoma herpesvirus (KSHV) establishes lifelong latency. The viral latency-associated nuclear antigen (LANA) promotes viral persistence by tethering the viral genome to cellular chromosomes and by participating in latent DNA replication. Recently, the structure of the LANA C-terminal DNA binding domain was solved and new cytoplasmic variants of LANA were discovered. We discuss how these findings contribute to our current view of LANA structure and assembly and of its role during viral persistence.

scholarly journals Rolling Circle Amplification is a high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

2020 ◽  
Author(s):  
Jeffrey M. Marano ◽  
Christina Chuong ◽  
James Weger-Lucarelli
Keyword(s):  
Cdna Clone ◽  
Plasmid Dna ◽  
Viral Genome ◽  
Traditional Approach ◽  
Rolling Circle Amplification ◽  
Infectious Cdna Clone ◽  
Cdna Clones ◽  
High Fidelity ◽  
Rolling Circle ◽  
Viral Yield

AbstractAlphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus, Eastern equine encephalitis virus, and the emerging Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology and to create effective vaccines. The traditional approach to rescuing virus from an infectious cDNA clone requires propagating large amounts of plasmids in bacteria, which can result in unwanted mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative to the bacterial-based plasmid platform that uses rolling circle amplification (RCA), an in vitro technology that amplifies plasmid DNA using only basic equipment. We demonstrate that the use of RCA to amplify plasmid DNA is comparable to the use of a midiprepped plasmid in terms of viral yield, albeit with a slight delay in virus recovery kinetics. RCA, however, has lower cost and time requirements and amplifies DNA with high fidelity and with no chance of unwanted mutations due to toxicity. We show that sequential RCA reactions do not introduce mutations into the viral genome and, thus, can replace the need for glycerol stocks or bacteria entirely. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.ImportanceThe development of infectious cDNA clones is critical to studying viral pathogenesis and for developing vaccines. The current method for propagating clones in bacteria is limited by the toxicity of the viral genome within the bacterial host, resulting in deleterious mutations in the viral genome, which can only be detected through whole-genome sequencing. These mutations can attenuate the virus, leading to lost time and resources and potentially confounding results. We have developed an alternative method of preparing large quantities of DNA that can be directly transfected to recover infectious virus without the need for bacteria by amplifying the infectious cDNA clone plasmid using rolling circle amplification (RCA). Our results indicate that viral rescue from an RCA product produces a viral yield equal to bacterial-derived plasmid DNA, albeit with a slight delay in replication kinetics. The RCA platform, however, is significantly more cost and time-efficient compared to traditional approaches. When the simplicity and costs of RCA are combined, we propose that a shift to an RCA platform will benefit the field of molecular virology and could have significant advantages for recombinant vaccine production.

scholarly journals Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance

2010 ◽  
Vol 84 (22) ◽  
pp. 11747-11760 ◽  
Author(s):  
Amélie Fradet-Turcotte ◽  
Cary Moody ◽  
Laimonis A. Laimins ◽  
Jacques Archambault
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Nuclear Export ◽  
Viral Genome ◽  
Cellular Proliferation ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Genome Maintenance ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.

scholarly journals Human Cytomegalovirus UL84 Insertion Mutant Defective for Viral DNA Synthesis and Growth

2004 ◽  
Vol 78 (19) ◽  
pp. 10360-10369 ◽  
Author(s):  
Yiyang Xu ◽  
Sylvia A. Cei ◽  
Alicia Rodriguez Huete ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Infectious Virus ◽  
Regulatory Protein ◽  
Insertion Mutant ◽  
Viral Dna ◽  
Transfected Cells

ABSTRACT Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent DNA replication, and evidence from transient transfection assays suggests that UL84 directly participates in DNA synthesis. In addition, because of its apparent interaction with IE2, UL84 is implicated as a possible regulatory protein. To address the role of UL84 in the context of the viral genome, we generated a recombinant HCMV bacterial artificial chromosome (BAC) construct that did not express the UL84 gene product. This construct, BAC-IN84/Ep, displayed a null phenotype in that it failed to produce infectious virus after transfection into human fibroblast cells, whereas a revertant virus readily produced viral plaques and, subsequently, infectious virus. Real-time quantitative PCR showed that BAC-IN84/Ep was defective for DNA synthesis in that no increase in the accumulation of viral DNA was observed in transfected cells. We were unable to complement BAC-IN84/Ep in trans; however, oriLyt-dependent DNA replication was observed by the cotransfection of UL84 and BAC-IN84/Ep. An analysis of viral mRNA by real-time PCR indicated that, even in the absence of DNA synthesis, all representative kinetic classes of genes were expressed in cells transfected with BAC-IN84/Ep. The detection of UL44 and IE2 by immunofluorescence in BAC-IN84/Ep-transfected cells showed that these proteins failed to partition into replication compartments, indicating that UL84 expression is essential for the formation of these proteins into replication centers within the context of the viral genome. These results show that UL84 provides an essential DNA replication function and influences the subcellular localization of other viral proteins.

scholarly journals Acetylation of E2 by P300 Mediates Topoisomerase Entry at the Papillomavirus Replicon

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Yanique Thomas ◽  
Elliot J. Androphy
Keyword(s):  
Dna Replication ◽  
Viral Genome ◽  
Adult Population ◽  
Specific Effect ◽  
The United States ◽  
Dna Helicase ◽  
Human Papillomaviruses ◽  
Host Cells ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACTHuman papillomavirus (HPV) E2 proteins are integral for the transcription of viral genes and the replication and maintenance of viral genomes in host cells. E2 recruits the viral DNA helicase E1 to the origin. A lysine (K111), highly conserved among almost all papillomavirus (PV) E2 proteins, is a target for P300 (EP300) acetylation and is critical for viral DNA replication (E. J. Quinlan, S. P. Culleton, S. Y. Wu, C. M. Chiang, et al., J Virol 87:1497–1507, 2013, https://doi.org/10.1128/JVI.02771-12; Y. Thomas and E. J. Androphy, J Virol 92:e01912-17, 2018, https://doi.org/10.1128/JVI.01912-17). Since the viral genome exists as a covalently closed circle of double-stranded DNA, topoisomerase 1 (Topo1) is thought to be required for progression of the replication forks. Due to the specific effect of K111 mutations on DNA unwinding (Y. Thomas and E. J. Androphy, J Virol 92:e01912-17, 2018, https://doi.org/10.1128/JVI.01912-17), we demonstrate that the E2 protein targets Topo1 to the viral origin, and this depends on acetylation of K111. The effect was corroborated by functional replication assays, in which higher levels of P300, but not its homolog CBP, caused enhanced replication with wild-type E2 but not the acetylation-defective K111 arginine mutant. These data reveal a novel role for lysine acetylation during viral DNA replication by regulating topoisomerase recruitment to the replication origin.IMPORTANCEHuman papillomaviruses affect an estimated 75% of the sexually active adult population in the United States, with 5.5 million new cases emerging every year. More than 200 HPV genotypes have been identified; a subset of them are linked to the development of cancers from these epithelial infections. Specific antiviral medical treatments for infected individuals are not available. This project examines the mechanisms that control viral genome replication and may allow the development of novel therapeutics.

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