scholarly journals The KSHV ORF20 Protein Interacts with the Viral Processivity Factor ORF59 and Promotes Viral Reactivation

2021 ◽  
Author(s):  
D. Hoffman ◽  
W. Rodriguez ◽  
D. Macveigh-Fierro ◽  
J. Miles ◽  
M. Muller
Keyword(s):  
Life Cycle ◽  
Dna Replication ◽  
Viral Proteins ◽  
Lytic Replication ◽  
Viral Reactivation ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
New Hosts ◽  
Processivity Factor

Kaposi’s Sarcoma-associated herpesvirus (KSHV) is a herpesvirus that induces lifelong infection, and as such, its lytic replication is carefully controlled to allow for efficient dissemination from its long-term reservoir and for the spread of the virus to new hosts. Viral DNA replication involves many host and viral proteins, coordinating both in time and space to successfully progress through the viral life cycle.

scholarly journals Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication

2015 ◽  
Vol 89 (9) ◽  
pp. 4980-4991 ◽  
Author(s):  
Elaine J. Gauson ◽  
Mary M. Donaldson ◽  
Edward S. Dornan ◽  
Xu Wang ◽  
Molly Bristol ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Viral Proteins ◽  
Future Generations ◽  
Cellular Proteins ◽  
Origin Of Replication ◽  
Viral Dna ◽  
Viral Origin ◽  
Viral Dna Replication ◽  
Human Papillomavirus 16

ABSTRACTTo replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination.IMPORTANCEHuman papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.

scholarly journals The KSHV ORF20 Protein Interacts With The Viral Processivity Factor ORF59 And Promote Viral Reactivation

2020 ◽  
Author(s):  
D. Hoffman ◽  
W. Rodriguez ◽  
D. Macveigh-Fierro ◽  
J. Miles ◽  
M. Muller
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Viral Proteins ◽  
Lytic Replication ◽  
Viral Reactivation ◽  
Wild Type Virus ◽  
Viral Gene ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Processivity Factor

AbstractUpon KSHV lytic reactivation, rapid and widespread amplification of viral DNA (vDNA) triggers significant nuclear reorganization. As part of this striking shift in nuclear architecture, viral replication compartments are formed as sites of lytic vDNA production along with remarkable spatial remodeling and relocalization of cellular and viral proteins. These viral replication compartments house several lytic gene products that coordinate viral gene expression, vDNA replication, and nucleocapsid assembly. The viral proteins and mechanisms that regulate this overhaul of the nuclear landscape during KSHV replication remain largely unknown. KSHV’s ORF20 is a widely conserved lytic gene among all herpesviruses suggesting it may have a fundamental contribution to the progression of herpesviral infection. Here, we utilized a promiscuous biotin ligase proximity labeling method to identify the proximal interactome of ORF20, which includes several replication-associated viral proteins, one of which is ORF59, the KSHV DNA processivity factor. Using co-immunoprecipitation and immunofluorescence assays, we confirmed the interaction between ORF20 and ORF59 and tracked the localization of both proteins to KSHV replication compartments. To further characterize the function of ORF20, we generated an ORF20-deficient KSHV and compared its replicative fitness relative to wild type virus. Virion production was significantly diminished in the ORF20-deficient virus as observed by supernatant transfer assays. Additionally, we tied this defect in viable virion formation to a reduction in viral late gene expression. Lastly, we observed an overall reduction in vDNA replication in the ORF20-deficient virus implying a key role for ORF20 in the regulation of lytic replication. Taken together, these results capture the essential role of KSHV ORF20 in progressing viral lytic infection by regulating vDNA replication alongside other crucial lytic proteins within KSHV replication compartments.

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.

scholarly journals The SWI/SNF Chromatin Regulator BRG1 Modulates the Transcriptional Regulatory Activity of the Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Mei-Tzu Su ◽  
Ya-Ting Wang ◽  
Yen-Ju Chen ◽  
Su-Fang Lin ◽  
Ching-Hwa Tsai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Viral Gene ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1. IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.

scholarly journals Herpes Simplex Virus DNA Synthesis Is Not a Decisive Regulatory Event in the Initiation of Lytic Viral Protein Expression in Neurons In Vivo during Primary Infection or Reactivation from Latency

2006 ◽  
Vol 80 (1) ◽  
pp. 38-50 ◽  
Author(s):  
N. M. Sawtell ◽  
R. L. Thompson ◽  
R. L. Haas
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Sensory Neurons ◽  
Viral Proteins ◽  
Lytic Cycle ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Simplex Virus

ABSTRACT The herpes simplex virus genome can enter a repressed transcriptional state (latency) in sensory neurons of the host nervous system. Although reduced permissiveness of the neuronal environment is widely accepted as a causal factor, the molecular pathway(s) directing and maintaining the viral genome in the latent state remains undefined. Over the past decade, the field has been strongly influenced by the observations of Kosz-Vnenchak et al., which have been interpreted to indicate that, in sensory neurons in vivo, a critical level of viral DNA synthesis within the neuron is required for sufficient viral immediate-early (IE) and early (E) gene expression (M. Kosz-Vnenchak, J. Jacobson, D. M. Coen, and D. M. Knipe, J. Virol. 67:5383-5393, 1993). The levels of IE and E genes are, in turn, thought to regulate the decision to enter the lytic cycle or latency. We have reexamined this issue using new strategies for in situ detection and quantification of viral gene expression in whole tissues. Our results using thymidine kinase-null and rescued mutants as well as wild-type strains in conjunction with viral DNA synthesis blockers demonstrate that (i) despite inhibition of viral DNA replication, many neurons express lytic viral proteins, including IE proteins, during acute infection in the ganglion; (ii) at early times postinoculation, the number of neurons expressing viral proteins in the ganglion is not reduced by inhibition of viral DNA replication; and (iii) following a reactivation stimulus, the numbers of neurons and apparent levels of lytic viral proteins, including IE proteins, are not reduced by inhibition of viral DNA replication. We conclude that viral DNA replication in the neuron per se does not regulate IE gene expression or entry into the lytic cycle.

scholarly journals Role of the Specific Interaction of UL112-113 p84 with UL44 DNA Polymerase Processivity Factor in Promoting DNA Replication of Human Cytomegalovirus

2010 ◽  
Vol 84 (17) ◽  
pp. 8409-8421 ◽  
Author(s):  
Young-Eui Kim ◽  
Jin-Hyun Ahn
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Specific Interaction ◽  
Terminal Region ◽  
Replication Proteins ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Carboxy Terminal Region ◽  
Processivity Factor ◽  
Carboxy Terminal

ABSTRACT The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.

scholarly journals Host AAA+ ATPase TER94 Plays Critical Roles in Building the Baculovirus Viral Replication Factory and Virion Morphogenesis

2020 ◽  
Vol 94 (6) ◽  
Author(s):  
Yimeng Li ◽  
Liangbo Hu ◽  
Tong Chen ◽  
Meng Chang ◽  
Fei Deng ◽  
...  
Keyword(s):  
Life Cycle ◽  
Dna Replication ◽  
Viral Replication ◽  
Protein Quality ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Early Proteins ◽  
Virion Morphogenesis

ABSTRACT TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis. IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.

scholarly journals Histone Deacetylase Inhibitors Promote Latent Adenovirus Reactivation from Tonsillectomy Specimens

2020 ◽  
Vol 94 (12) ◽  
Author(s):  
Lingling Wang ◽  
Mengyu Zhang ◽  
Jingjing Li ◽  
Guang Yang ◽  
Qun Huang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Young Children ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Viral Reactivation ◽  
Early Gene ◽  
Gene Promoters ◽  
Compound Library ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT Adenovirus (HAdV) infection is a common cause of illness among young children, immunocompromised patients, and transplant recipients. The majority of HAdV infections are self-limited, but recurring infection is frequently encountered in young children and may require hospitalization. In this study, we surveyed the presence of HAdV in tonsillectomy samples and investigated epigenetic conditions that contributed to HAdV reactivation. HAdV DNA was detected from 86.7% donors. The lymphocytes isolated from the samples failed to produce infectious HAdV after incubation, suggesting the viruses remained in a latent status. To determine whether epigenetic factors played a role in HAdV reactivation, isolated lymphocytes were treated with a small compound library. Viral DNA replication and infectious HAdV production were assayed by PCR and by a secondary infection assay. We identified several compounds, mainly pan- and selective histone deacetylase (HDAC) inhibitors, which showed activity to reactivate HAdV from latency. The viruses were isolated and were determined as species C HAdV. Using a model of HAdV lytic infection, we showed that the compounds promoted histone-3 acetylation and association with viral early gene promoters. In addition to demonstrate the palatine tonsils as a reservoir of latent HAdV, this study uncovers a critical role of histone acetylation in HAdV reactivation, linking HAdV latency to recurrent HAdV infection. IMPORTANCE Respiratory tract infection by adenoviruses is among the most common diseases in children, attributing to approximately 20% of hospitalizations of children with acute respiratory infection (ARI). Adenovirus transmits by direct contact, but recurrent infection is common. Ever since its isolation, adenovirus has been known to have the ability to establish persistent or latent infection. We found 87.7% tonsillectomy specimens contained detectable amounts of adenoviral DNA. Isolated lymphocytes did not produce infectious adenoviruses without stimulation. By screening an epigenetic informer compound library, we identified several histone deacetylase inhibitors that promoted adenovirus reactivation that was evidenced by increased viral DNA replication and production of infectious viruses. The human tonsils are covered with bacterial pathogens that may utilize pathogen-associated pattern molecules or metabolites to cause epigenetic activation and proinflammatory gene transcription, which may lead to viral reactivation from latency. The study shows that recurrent adenovirus infection could arise from reactivation of residing virus from previous infections.

scholarly journals Biology of Ovine Adenovirus Infection of Nonpermissive Cells

2002 ◽  
Vol 76 (21) ◽  
pp. 10882-10893 ◽  
Author(s):  
Daniel Kümin ◽  
Christian Hofmann ◽  
Michael Rudolph ◽  
Gerald W. Both ◽  
Peter Löser
Keyword(s):  
Dna Replication ◽  
Early Stage ◽  
Virus Production ◽  
Adenovirus Type ◽  
Cell Types ◽  
Viral Proteins ◽  
Vector System ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Wide Range

ABSTRACT Nonhuman adenoviruses, including those of the genus Atadenovirus, have the potential to serve as vectors for vaccine and gene therapy applications in humans, since they are resistant to preexisting immunity induced by human adenoviruses in the majority of the population. In this study, we elucidate the outcome of infection by ovine adenovirus type 7 isolate 287 (OAdV) of several nonovine cell types. We show here that OAdV infects a wide range of nonovine cells but is unable to complete its replication cycle in any of them. In nonovine, nonfibroblast cells, viral replication is blocked at an early stage before the onset of, or early in, DNA replication. Some fibroblasts, on the other hand, allow viral DNA replication but block virus production at a later stage during or after the translation of late viral proteins. Late viral proteins are expressed in cells where viral DNA replication takes place, albeit at a reduced level. Significantly, late proteins are not properly processed, and their cellular distribution differs from that observed in infected ovine cells. Thus, our results clearly show that OAdV infection of all nonovine cells tested is abortive even if significant viral DNA replication occurs. These findings have significant positive implications with respect to the safety of the vector system and its future use in humans.

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