Multiloci Manipulation of Baculovirus Genome Reveals the Pivotal Role of Homologous Regions in Viral DNA Replication, Progeny Production, and Enhancing Transcription

ABSTRACT The oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus, also identified as human herpesvirus 8, contains genes producing proteins that control transcription and influence cell signaling. Open reading frame 36 (ORF36) of this virus encodes a serine/threonine protein kinase, which is designated the viral protein kinase (vPK). Our recent efforts to elucidate the role of vPK in the viral life cycle have focused on identifying viral protein substrates and determining the effects of vPK-mediated phosphorylation on specific steps in viral replication. The vPK gene was transcribed into 4.2-kb and 3.6-kb mRNAs during the early and late phases of viral reactivation. vPK is colocalized with viral DNA replication/transcription compartments as marked by a polymerase processivity factor, and K-bZIP, a protein known to bind the viral DNA replication origin (Ori-Lyt) and to regulate viral transcription. The vPK physically associated with and strongly phosphorylated K-bZIP at threonine 111, a site also recognized by the cyclin-dependent kinase Cdk2. Both K-bZIP and vPK were corecruited to viral promoters targeted by K-bZIP as well as to the Ori-Lyt region. Phosphorylation of K-bZIP by vPK had a negative impact on K-bZIP transcription repression activity. The extent of posttranslational modification of K-bZIP by sumoylation, a process that influences its repression function, was decreased by vPK phosphorylation at threonine 111. Our data thus identify a new role of vPK as a modulator of viral transcription.

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Baculovirus Infection Raises the Level of TATA-Binding Protein That Colocalizes with Viral DNA Replication Sites

Journal of Virology ◽

10.1128/jvi.76.21.11123-11127.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 11123-11127 ◽

Cited By ~ 11

Author(s):

Ilja Quadt ◽

Daniela Mainz ◽

Ruud Mans ◽

Andreas Kremer ◽

Dagmar Knebel-Mörsdorf

Keyword(s):

Dna Replication ◽

Rna Polymerase ◽

Binding Protein ◽

Tata Binding Protein ◽

Viral Transcription ◽

Mrna Levels ◽

Viral Dna ◽

Viral Dna Replication ◽

Baculovirus Infection

ABSTRACT During the infection cycle of Autographa californica multicapsid nuclear polyhedrosis virus, the TATA-binding protein (TBP) of the insect host cell likely participates in early viral transcription, which is mediated by the host RNA polymerase II. However, the role of TBP in late and very late viral transcription, which is accomplished by an alpha-amanitin-resistant RNA polymerase, is unclear. We observed a dramatic increase of TBP protein during the late phases of infection. TBP mRNA levels, however, were not coordinately increased. Indirect-immunofluorescence studies revealed a nuclear redistribution of TBP during infection. After labeling of viral replication centers with bromodeoxyuridine (BrdU), costaining of TBP and BrdU showed that TBP localized to viral DNA replication centers. These results suggest a putative role of TBP during late viral transcription, which may occur in close proximity to viral DNA replication.

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Dissecting the role of the ϕ29 terminal protein DNA binding residues in viral DNA replication

Nucleic Acids Research ◽

10.1093/nar/gkv127 ◽

2015 ◽

Vol 43 (5) ◽

pp. 2790-2801 ◽

Cited By ~ 1

Author(s):

Isabel Holguera ◽

Daniel Muñoz-Espín ◽

Margarita Salas

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Viral Dna ◽

Terminal Protein ◽

Viral Dna Replication ◽

Binding Residues

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Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

Journal of Virology ◽

10.1128/jvi.02177-17 ◽

2018 ◽

Vol 92 (7) ◽

Cited By ~ 4

Author(s):

Dongcheng Liu ◽

Yan Wang ◽

Yan Yuan

Keyword(s):

Dna Replication ◽

Noncoding Rna ◽

Rna Binding ◽

De Novo ◽

Binding Protein ◽

Rna Binding Protein ◽

Noncoding Rnas ◽

Viral Dna ◽

Viral Dna Replication

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation andde novoinfection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UVcross-linking andimmunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication duringde novoinfection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication duringde novoinfection.IMPORTANCEGenomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.

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