Contribution of bovine papillomavirus type 1 E1 protein residue 48 to replication function

Herpes simplex virus type 1 (HSV-1) ICP0 directs the degradation of cellular proteins associated with nuclear structures called ND10, a function thought to be closely associated with its broad transactivating activity. Roscovitine (Rosco), an inhibitor of cyclin-dependent kinases (cdks), inhibits the replication of HSV-1, HSV-2, human cytomegalovirus, varicella-zoster virus, and human immunodeficiency virus type 1 by inhibiting specific steps or activities of viral regulatory proteins, indicating the broad and pleiotropic effects that cdks have on the replication of these viruses. We previously demonstrated that Rosco inhibits the transactivating activity of ICP0. In the present study, we asked whether Rosco also affects the ability of ICP0 to direct the degradation of ND10-associated proteins. For this purpose, WI-38 cells treated with cycloheximide (CHX) were mock infected or infected with wild-type HSV-1 or an ICP0؊ mutant (7134). After release from the CHX block, the infections were allowed to proceed for 2 h in the presence or absence of Rosco at a concentration known to inhibit ICP0's transactivating activity. The cells were then examined for the presence of ICP0 and selected ND10-associated antigens (promyelocytic leukemia antigen [PML], sp100, hDaxx, and NDP55) by immunofluorescence. Staining for the ND10-associated antigens was detected in <20% of KOS-infected cells in the presence or absence of Rosco, demonstrating that Rosco-sensitive kinases are not required for ICP0's ability to direct the dispersal or degradation of these antigens. In contrast, >90% of 7134-and mock-infected cells stained positive for all ND10-associated antigens in the presence or absence of Rosco. Similar results were obtained with a non-ND10-associated antigen, DNA-PK cs , a known target of ICP0-directed degradation. The results of the PML and DNA-PK cs immunofluorescence studies correlated with a decrease in the levels of these proteins as determined by Western blotting. Thus, the differential requirement for Rosco-sensitive cdk activities distinguishes ICP0's ability to direct the dispersal or degradation of cellular proteins from its transactivating activity.The molecular mechanisms responsible for the activation of herpes simplex virus (HSV) gene expression during productive infection and reactivation from latency are not well understood. Available evidence indicates, however, that the functions of viral regulatory proteins that activate viral-gene expression are themselves activated by cell cycle regulatory proteins. For example, two cellular proteins, HCF (36) and TAF250 (84), whose expression and activities are cell cycle associated, activate the functions of two viral regulatory proteins, VP16 (36) and ICP4 (12), respectively, and cyclin-dependent kinases (cdks), which drive the cell cycle, are required for the synthesis and activities of HSV immediate-early (IE) regulatory proteins (75-77). Notably, inhibitors of cdks have been shown to inhibit the replication of HSV type 1 (HSV-1) (74), HSV-2 (74), human cytomegaloviru...

Section: Discussionmentioning

confidence: 99%

The Differential Requirement for Cyclin-Dependent Kinase Activities Distinguishes Two Functions of Herpes Simplex Virus Type 1 ICP0

Davido

Zagorski

Maul

et al. 2003

“…Besides threonine 102 and serine 109 located at the NLS sequence, the BPV1 E1 protein is phosphorylated or predicted to be phosphorylated at several additional amino acid residues by the action of different host kinases (61). There are two reported CKII sites, serines 48 and 584 (29,36,37), and two putative CDK sites, threonine 126 and serine 283, in addition to the known CDK site at threonine 102. To test if phosphorylation at these other four sites might regulate E1 nuclear import, we examined the importin ␣ binding capacities of three additional sets of pseudophosphorylation mutants: CKII (S48D/S584D), CDK (T102D/T126D/S283D), and ALL (S48D/T102D/T126D/S283D/S584D) (51).…”

Section: Bpv1 E1 Protein Binds To Importins ␣3 ␣4mentioning

confidence: 99%

“…To meet these replication requirements, the E1 protein is regulated not only at the expression level (44) but also by posttranslational modifications such as sumoylation (48, 49), ubiquitination (34, 38), and phosphorylation. Bovine papillomavirus type 1 (BPV1) E1 protein is phosphorylated at multiple sites and by different kinases, including serines 48 and 584 by CKII (29,36,37), threonine 102 by p34 cdc2 (9,30), and serine 109 by protein kinase C (PKC) (64). Mutational analysis indicates that changes in these residues can affect viral replication, but in most cases, mechanistic details are lacking.…”

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confidence: 99%

Nuclear Import of Bovine Papillomavirus Type 1 E1 Protein Is Mediated by Multiple Alpha Importins and Is Negatively Regulated by Phosphorylation near a Nuclear Localization Signal

Bian

Rosas-Acosta

et al. 2007

Self Cite

Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral E1 protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the E1 protein from bovine papillomavirus type 1 (BPV1). Using an in vitro binding assay, we determined that the E1 protein interacted with importins ␣3, ␣4, and ␣5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified E1 protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin ␣3, ␣4, or ␣5 and other necessary import factors. We also observed that in vitro binding of E1 protein to all three ␣ importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated E1 protein failed to enter the nucleus of digitonin-permeabilized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells, while the corresponding alanine replacement mutant displayed the same cellular location pattern as wild-type E1 protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin ␣ and suggest that E1 phosphorylation by host cell kinases plays a regulatory role in modulating E1 nucleocytoplasmic localization. This phosphoregulation of nuclear E1 protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation.Papillomaviruses are the etiological agents involved in several human cancers such as cervical cancer, anogenital cancer, skin cancer, and cancers of the oral cavity, the larynx, and the esophagus (68). In addition to their importance in clinical disease, papillomaviruses have provided a valuable model system for analyzing the mechanisms regulating eukaryotic DNA replication. The viral E1 protein is the largest open reading frame and is highly conserved among all papillomaviruses, maintaining its size, amino acid composition, and location in the viral genome with respect to other early genes. The E1 protein is expressed during the early stage of virus infection in order to maintain the viral DNA as an episome. The multifunctional E1 protein recognizes and binds to the viral origin of replication in combination with the viral protein E2, recruits host cell replication proteins to the origin, and initiates DNA replication via its ATP-dependent helicase activity (60, 61).Papillomavirus infection is established in the basal layer of the epithelium, and the complex viral life cycle is coordinated with the differentiation state of the epithelium (13, 14). There are three distinct modes of viral DNA replication: (i) transient amplification, which occurs immediately upon ...

“…Upon entry of a host cell by wounding or abrasion, BPV replicates its small 8-kb genome to a multicopy episome which is maintained at steady-state levels of up to several hundred copies per cell (14). After a steadystate level of replication has been achieved, the viral DNA then replicates approximately once during each host cell cycle and is likely regulated by cell cycle controls (8,18,21). In order to initiate viral DNA replication, the E1 protein forms a multimeric complex with the E2 protein, the viral origin of replication, and several host cell factors (2,6,8,13,15,26,28,33).…”

mentioning

confidence: 99%

“…It is a multifunctional 68-kDa phosphoprotein which cooperates with the E2 protein to bind sequence-specifically to its cognate E1 binding element in the viral origin, facilitates origin DNA unwinding by acting as an ATP-dependent helicase, recruits the host cell DNA polymerase ␣-primase complex, which then begins de novo DNA synthesis, and interacts with regulatory host cell factors such as cyclin E (8, 18, 23, 28, 32, 38). In addition, the phosphorylation state of E1 has been suggested as a regulatory device and link to the cell cycle control apparatus, while sumoylation is required for nuclear localization (8,18,19,21,24,25,39).…”

mentioning

confidence: 99%

Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein

West

Flanery

Woytek

et al. 2001

Self Cite

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1 [179][180][181][182][183][184][185][186][187][188][189][190][191] ) and HR3 (E1 241-252 ), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 ␣-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).Papillomaviruses are members of the papovavirus family and comprise both human and animal strains. In addition to causing common cutaneous warts, papillomaviruses can induce various skin and mucosal lesions (35,36,39). Some of these lesions may progress to malignant carcinomas, depending on the particular viral strain involved as well as environmental factors. Malignancies which are highly associated with papillomavirus infection include laryngeal, cervical, and other anogenital cancers, and it is noteworthy that human papillomavirus (HPV) types 16, 18, 31, 33, and 45 pose an especially high risk for females, as they are estimated to be present in at least ...