Conditional repression of the E2 transcription unit in E1-E3-deleted adenovirus vectors is correlated with a strong reduction in viral DNA replication and late gene expression in vitro.

1997 ◽  
Vol 71 (4) ◽  
pp. 3307-3311 ◽  
Author(s):  
K Rittner ◽  
H Schultz ◽  
A Pavirani ◽  
M Mehtali
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Transcription Unit ◽  
Late Gene ◽  
Late Gene Expression ◽  
Strong Reduction ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Adenovirus Vectors

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 Regulation of Viral Intermediate Gene Expression by the Vaccinia Virus B1 Protein Kinase

2001 ◽  
Vol 75 (9) ◽  
pp. 4048-4055 ◽  
Author(s):  
Gerald R. Kovacs ◽  
Nikos Vasilakis ◽  
Bernard Moss
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Protein Kinase ◽  
Vaccinia Virus ◽  
Gene Transcription ◽  
Late Gene ◽  
Nonpermissive Temperature ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The B1 gene of vaccinia virus encodes a serine/threonine protein kinase that is expressed early after infection. Under nonpermissive conditions, temperature-sensitive mutants (ts2 andts25) that map to B1 fail to efficiently replicate viral DNA. Our goal was to extend studies on the function of B1 by determining if the kinase is required for intermediate or late gene expression, two events that ordinarily depend on viral DNA replication. First, we established that early viral gene expression occurred at the nonpermissive temperature. By using a transfection procedure that circumvents the viral DNA replication requirement, we found that reporter genes regulated by an intermediate promoter were transcribed only under conditions permissive for expression of active B1. To assay late gene expression, the T7 RNA polymerase gene was inserted into the genome of ts25 to form ts25/T7. A DNA replication-independent late gene transcription system was established by cotransfecting plasmids containing T7 promoter-driven late gene transcription factors and a late promoter reporter gene intots25/T7-infected cells. Late genes, unlike intermediate genes, were expressed at the nonpermissive temperature. Last, we showed that overexpression of B1 stimulated intermediate but inhibited late gene expression in cells infected with wild-type virus.

scholarly journals A Dominant-Negative Herpesvirus Protein Inhibits Intranuclear Targeting of Viral Proteins: Effects on DNA Replication and Late Gene Expression

2000 ◽  
Vol 74 (21) ◽  
pp. 10122-10131 ◽  
Author(s):  
Elizabeth E. McNamee ◽  
Travis J Taylor ◽  
David M. Knipe
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Viral Replication ◽  
Gene Transcription ◽  
Dominant Negative ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Late Genes ◽  
Infected Cells

ABSTRACT The d105 dominant-negative mutant form of the herpes simplex virus 1 (HSV-1) single-stranded DNA-binding protein, ICP8 (d105 ICP8), inhibits wild-type viral replication, and it blocks both viral DNA replication and late gene transcription, although to different degrees (M. Gao and D. M. Knipe, J. Virol. 65:2666–2675, 1991; Y. M. Chen and D. M. Knipe, Virology 221:281–290, 1996). We demonstrate here that this protein is also capable of preventing the formation of intranuclear prereplicative sites and replication compartments during HSV infection. We defined three patterns of ICP8 localization using indirect immunofluorescence staining of HSV-1-infected cells: large replication compartments, small compartments, and no specific intranuclear localization of ICP8. Cells that form large replication compartments replicate viral DNA and express late genes. Cells that form small replication compartments replicate viral DNA but do not express late genes, while cells without viral replication compartments are incapable of both DNA replication and late gene expression. The d105 ICP8 protein blocks formation of prereplicative sites and large replication compartments in 80% of infected cells and formation of large replication compartments in the remaining 20% of infected cells. The phenotype ofd105 suggests a correlation between formation of large replication compartments and late gene expression and a role for intranuclear rearrangement of viral DNA and bound proteins in activation of late gene transcription. Thus, these results provide evidence for specialized machinery for late gene expression within replication compartments.

scholarly journals An Epigenetic Journey: Epstein-Barr Virus Transcribes Chromatinized and Subsequently Unchromatinized Templates during Its Lytic Cycle

2019 ◽  
Vol 93 (8) ◽  
Author(s):  
Adityarup Chakravorty ◽  
Bill Sugden ◽  
Eric C. Johannsen
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Epstein Barr Virus ◽  
Dna Amplification ◽  
Lytic Cycle ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTThe Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication incis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

scholarly journals The HIV Integrase Inhibitor Raltegravir Inhibits Felid Alphaherpesvirus 1 Replication by Targeting both DNA Replication and Late Gene Expression

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Matthew R. Pennington ◽  
Ian E. H. Voorhees ◽  
Heather M. Callaway ◽  
Shannon D. Dehghanpir ◽  
Joel D. Baines ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Drug Application ◽  
Integrase Inhibitor ◽  
Late Gene ◽  
Drug Resistant ◽  
Hiv Integrase ◽  
Late Gene Expression

ABSTRACTAlphaherpesvirus-associated ocular infections in humans caused by human alphaherpesvirus 1 (HHV-1) remain challenging to treat due to the frequency of drug application required and the potential for the selection of drug-resistant viruses. Repurposing on-the-market drugs is a viable strategy to accelerate the pace of drug development. It has been reported that the human immunodeficiency virus (HIV) integrase inhibitor raltegravir inhibits HHV-1 replication by targeting the DNA polymerase accessory factor and limits terminase-mediated genome cleavage of human betaherpesvirus 5 (HHV-5). We have previously shown, bothin vitroandin vivo, that raltegravir can also inhibit the replication of felid alphaherpesvirus 1 (FeHV-1), a common ocular pathogen of cats with a pathogenesis similar to that of HHV-1 ocular disease. In contrast to what was reported for HHV-1, we were unable to select for a raltegravir-resistant FeHV-1 strain in order to define any basis for drug action. A candidate-based approach to explore the mode of action of raltegravir against FeHV-1 showed that raltegravir did not impact FeHV-1 terminase function, as described for HHV-5. Instead, raltegravir inhibited DNA replication, similarly to HHV-1, but by targeting the initiation of viral DNA replication rather than elongation. In addition, we found that raltegravir specifically repressed late gene expression independently of DNA replication, and both activities are consistent with inhibition of ICP8. Taken together, these results suggest that raltegravir could be a valuable therapeutic agent against herpesviruses.IMPORTANCEThe rise of drug-resistant herpesviruses is a longstanding concern, particularly among immunocompromised patients. Therefore, therapies targeting viral proteins other than the DNA polymerase that may be less likely to lead to drug-resistant viruses are urgently needed. Using FeHV-1, an alphaherpesvirus closely related to HHV-1 that similarly causes ocular herpes in its natural host, we found that the HIV integrase inhibitor raltegravir targets different stages of the virus life cycle beyond DNA replication and that it does so without developing drug resistance under the conditions tested. This shows that the drug could provide a viable strategy for the treatment of herpesvirus infections.

scholarly journals Transcription Program of Red Sea Bream Iridovirus as Revealed by DNA Microarrays

2005 ◽  
Vol 79 (24) ◽  
pp. 15151-15164 ◽  
Author(s):  
Dang Thi Lua ◽  
Motoshige Yasuike ◽  
Ikuo Hirono ◽  
Takashi Aoki
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Red Sea ◽  
Sea Bream ◽  
Protein Synthesis Inhibitor ◽  
Red Sea Bream ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Transcription Program

ABSTRACT Red sea bream iridovirus (RSIV) has been identified as the causative agent of a serious disease in red sea bream and at least 30 other marine fish species. We developed a viral DNA microarray containing 92 putative open reading frames of RSIV to monitor the viral gene transcription program over the time course of an in vitro infection and to classify RSIV transcripts into temporal kinetic expression classes. The microarray analysis showed that viral genes commenced expression as early as 3 h postinfection (p.i.) and this was followed by a rapid escalation of gene expression from 8 h p.i. onwards. Based on the expression of some enzymes associated with viral DNA replication, the DNA replication of RSIV appeared to begin at around 8 h p.i. in infected cells in vitro. Using a de novo protein synthesis inhibitor (cycloheximide) and a viral DNA replication inhibitor (phosphonoacetic acid), 87 RSIV transcripts could be classified into three temporal kinetic classes: nine immediate-early (IE), 40 early (E), and 38 late (L) transcripts. The gene expression of RSIV occurred in a temporal kinetic cascade with three stages (IE, E, and L). Although the three classes of transcripts were distributed throughout the RSIV genome, E transcripts appeared to cluster in at least six discrete regions and L transcripts appeared to originate from seven discrete regions. The microarray data were statistically confirmed by using a t test, and were also clustered into groups based on similarity in the gene expression patterns by using a cluster program.

scholarly journals ORF18 Is a Transfactor That Is Essential for Late Gene Transcription of a Gammaherpesvirus

2006 ◽  
Vol 80 (19) ◽  
pp. 9730-9740 ◽  
Author(s):  
Vaithilingaraja Arumugaswami ◽  
Ting-Ting Wu ◽  
DeeAnn Martinez-Guzman ◽  
Qingmei Jia ◽  
Hongyu Deng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Lytic Replication ◽  
Early Gene ◽  
Late Gene ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Murine Gammaherpesvirus ◽  
Late Genes

ABSTRACT Lytic replication of the tumor-associated human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus has important implications in pathogenesis and tumorigenesis. Herpesvirus lytic genes have been temporally classified as exhibiting immediate-early (IE), early, and late expression kinetics. Though the regulation of IE and early gene expression has been studied extensively, very little is known regarding the regulation of late gene expression. Late genes, which primarily encode virion structural proteins, require viral DNA replication for their expression. We have identified a murine gammaherpesvirus 68 (MHV-68) early lytic gene, ORF18, essential for viral replication. ORF18 is conserved in both beta- and gammaherpesviruses. By generating an MHV-68 ORF18-null virus, we characterized the stage of the virus lytic cascade that requires the function of ORF18. Gene expression profiling and quantitation of viral DNA synthesis of the ORF18-null virus revealed that the expression of early genes and viral DNA replication were not affected; however, the transcription of late genes was abolished. Hence, we have identified a gammaherpesvirus-encoded factor essential for the expression of late genes independently of viral DNA synthesis.

scholarly journals Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Danielle E. Lyons ◽  
Kuan-Ping Yu ◽  
Jason A. Vander Heiden ◽  
Lee Heston ◽  
Dirk P. Dittmer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Replication ◽  
Lytic Cycle ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Barr Virus ◽  
Late Genes ◽  
S Proteins

ABSTRACTEpstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator.IMPORTANCEMutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

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