scholarly journals Distinct Roles of the Adenovirus E4 ORF3 Protein in Viral DNA Replication and Inhibition of Genome Concatenation

2003 ◽  
Vol 77 (9) ◽  
pp. 5295-5304 ◽  
Author(s):  
Jared D. Evans ◽  
Patrick Hearing
Keyword(s):  
Dna Replication ◽  
Viral Infection ◽  
Point Mutations ◽  
Virus Production ◽  
Strand Break ◽  
Cell Protein ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Orf3 Protein

ABSTRACT Adenovirus early proteins E4 ORF3 and E4 ORF6 have complementary functions during viral infection. Both proteins facilitate efficient viral DNA replication, late protein expression, and prevention of concatenation of viral genomes. Additionally, E4 ORF6 is involved in the shutoff of the host cell protein synthesis through its interaction with the E1B 55K protein. This complex also leads to the degradation of p53. A unique function of E4 ORF3 is the reorganization of nuclear structures known as PML oncogenic domains (PODs). The function of these domains is unclear, but PODs have been implicated in a number of important cellular processes, including transcriptional regulation, apoptosis, transformation, and response to interferon. The goal of this study was to determine the functional significance of the reorganization of PODs by E4 ORF3. Point mutations were made in the E4 ORF3 gene. These mutants were recombined into a virus lacking E4 ORF6 and expressed under the control of the natural virus E4 promoter. The panel of mutant viruses was used to investigate the role of E4 ORF3 during the course of the viral infection program. One of the mutant viruses exhibited aberrant reorganization of PODs and had a severe defect in viral DNA replication, thus leading to a dramatic decrease in virus production. A number of mutants accumulated viral DNA and infectious virus particles to wild-type levels but showed significant viral genome concatenation. These data show that E4 ORF3 is a multifunctional protein and that a specific rearrangement of nuclear PML domains is coupled to efficient viral DNA replication. This function is distinct from the role of E4 ORF3 in the regulation of virus genome concatenation via inhibition of cellular double-strand break repair.

scholarly journals HIV Integrase Inhibitors Block Replication of Alpha-, Beta-, and Gammaherpesviruses

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Zhipeng Yan ◽  
Kevin F. Bryant ◽  
Sean M. Gregory ◽  
Magdalena Angelova ◽  
David H. Dreyfus ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rnase H ◽  
Cell Protein ◽  
Hiv Integrase ◽  
Integrase Inhibitors ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Hiv Integrase Inhibitors ◽  
Hsv 1

ABSTRACTThe catalytic site of the HIV integrase is contained within an RNase H-like fold, and numerous drugs have been developed that bind to this site and inhibit its activity. Herpes simplex virus (HSV) encodes two proteins with potential RNase H-like folds, the infected cell protein 8 (ICP8) DNA-binding protein, which is necessary for viral DNA replication and exhibits recombinase activityin vitro, and the viral terminase, which is essential for viral DNA cleavage and packaging. Therefore, we hypothesized that HIV integrase inhibitors might also inhibit HSV replication by targeting ICP8 and/or the terminase. To test this, we evaluated the effect of 118-D-24, a potent HIV integrase inhibitor, on HSV replication. We found that 118-D-24 inhibited HSV-1 replication in cell culture at submillimolar concentrations. To identify more potent inhibitors of HSV replication, we screened a panel of integrase inhibitors, and one compound with greater anti-HSV-1 activity, XZ45, was chosen for further analysis. XZ45 significantly inhibited HSV-1 and HSV-2 replication in different cell types, with 50% inhibitory concentrations that were approximately 1 µM, but exhibited low cytotoxicity, with a 50% cytotoxic concentration greater than 500 µM. XZ45 blocked HSV viral DNA replication and late gene expression. XZ45 also inhibited viral recombination in infected cells and ICP8 recombinase activityin vitro. Furthermore, XZ45 inhibited human cytomegalovirus replication and induction of Kaposi’s sarcoma herpesvirus from latent infection. Our results argue that inhibitors of enzymes with RNase H-like folds may represent a general antiviral strategy, which is useful not only against HIV but also against herpesviruses.IMPORTANCEThe herpesviruses cause considerable morbidity and mortality. Nucleoside analogs have served as effective antiviral agents against the herpesviruses, but resistance can arise through viral mutation. Second-line anti-herpes drugs have limitations in terms of pharmacokinetic properties and/or toxicity, so there is a great need for additional drugs for treatment of herpesviral infections. This study showed that the HIV integrase inhibitors also block herpesviral infection, raising the important potential of a new class of anti-herpes drugs and the prospect of drugs that combat both HIV and the herpesviruses.

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 Antiviral Properties of the LSD1 Inhibitor SP-2509

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Mitchell R. Harancher ◽  
Jessica E. Packard ◽  
Shane P. Cowan ◽  
Neal A. DeLuca ◽  
Jill A. Dembowski
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Dna Replication ◽  
Viral Replication ◽  
Virus Production ◽  
Coupled Processes ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Pol Ii ◽  
Hsv 1

ABSTRACT Lysine-specific demethylase 1 (LSD1) targets cellular proteins, including histone H3, p53, E2F, and Dnmt1, and is involved in the regulation of gene expression, DNA replication, the cell cycle, and the DNA damage response. LSD1 catalyzes demethylation of histone H3K9 associated with herpes simplex virus 1 (HSV-1) immediate early (IE) promoters and is necessary for IE gene expression, viral DNA replication, and reactivation from latency. We previously found that LSD1 associates with HSV-1 replication forks and replicating viral DNA, suggesting that it may play a direct role in viral replication or coupled processes. We investigated the effects of the LSD1 inhibitor SP-2509 on the HSV-1 life cycle. Unlike previously investigated LSD1 inhibitors tranylcypromine (TCP) and OG-L002, which covalently attach to the LSD1 cofactor flavin adenine dinucleotide (FAD) to inhibit demethylase activity, SP-2509 has previously been shown to inhibit LSD1 protein-protein interactions. We found that SP-2509 does not inhibit HSV-1 IE gene expression or transcription factor and RNA polymerase II (Pol II) association with viral DNA prior to the onset of replication. However, SP-2509 does inhibit viral DNA replication, late gene expression, and virus production. We used EdC labeling of nascent viral DNA to image aberrant viral replication compartments that form in the presence of SP-2509. Treatment resulted in the formation of small replication foci that colocalize with replication proteins but are defective for Pol II recruitment. Taken together, these data highlight a potential new role for LSD1 in the regulation of HSV-1 DNA replication and gene expression after the onset of DNA replication. IMPORTANCE Treatment of HSV-1-infected cells with SP-2509 blocked viral DNA replication, gene expression after the onset of DNA replication, and virus production. These data support a potential new role for LSD1 in the regulation of viral DNA replication and successive steps in the virus life cycle, and further highlight the promising potential to utilize LSD1 inhibition as an antiviral approach.

Immunological characterization of the role of adenovirus terminal protein in viral DNA replication

Virology ◽  
1983 ◽  
Vol 131 (2) ◽  
pp. 287-295 ◽  
Author(s):  
A.W.M. Rijnders ◽  
B.G.M. Van Bergen ◽  
P.C. Van Der Vliet ◽  
J.S. Sussenbach
Keyword(s):  
Dna Replication ◽  
Viral Dna ◽  
Terminal Protein ◽  
Viral Dna Replication ◽  
Immunological Characterization
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