scholarly journals The SMC5/6 Complex Interacts with the Papillomavirus E2 Protein and Influences Maintenance of Viral Episomal DNA

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Peris Bentley ◽  
Min Jie Alvin Tan ◽  
Alison A. McBride ◽  
Elizabeth A. White ◽  
Peter M. Howley
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Protein Interactions ◽  
Persistent Infection ◽  
Viral Genome ◽  
Host Protein ◽  
Cellular Proteins ◽  
Genome Maintenance ◽  
Viral Dna ◽  
E2 Protein

ABSTRACTThe papillomavirus E2 protein executes numerous essential functions related to viral transcription, replication of viral DNA, and viral genome maintenance. Because E2 lacks enzymatic activity, many of these functions are mediated by interactions with host cellular proteins. Unbiased proteomics approaches have successfully identified a number of E2-host protein interactions. We have extended such studies and have identified and validated the cellular proteins structural maintenance of chromosome 5 (SMC5) and SMC6 as interactors of the viral E2 protein. These two proteins make up the core components of the SMC5/6 complex. The SMC5/6 complex is a member of the conserved structural maintenance of chromosomes (SMC) family of proteins, which are essential for genome maintenance. We have examined the role of SMC5/6 in various E2 functions. Our data suggest that SMC6 is not required for E2-mediated transcriptional activation, E1/E2-mediated transient replication, or differentiation-dependent amplification of viral DNA. Our data, however, suggest a role for SMC5/6 in viral genome maintenance.IMPORTANCEThe high-risk human papillomaviruses (HPVs) are the etiological cause of cervical cancer and the most common sexually transmitted infection. While the majority of infections may be asymptomatic or cause only benign lesions, persistent infection with the oncogenic high-risk HPV types may lead to serious diseases, such as cervical cancer, anogenital carcinoma, or head and neck oropharyngeal squamous cell carcinoma. The identification of virus-host protein interactions provides insights into the mechanisms of viral DNA persistence, viral genome replication, and cellular transformation. Elucidating the mechanism of early events in the virus replication cycle as well as of integration of viral DNA into host chromatin may present novel antiviral strategies and targets for counteracting persistent infection. The E2 protein is an important viral regulatory protein whose functions are mediated through interactions with host cell proteins. Here we explore the interaction of E2 with SMC5/6 and the functional consequences.

scholarly journals Virus-host protein-protein interactions between human papillomavirus 16 E6 A1 and D2/D3 sub-lineages: variances and similarities

2020 ◽  
Author(s):  
Guillem Dayer ◽  
Mehran L. Masoom ◽  
Melissa Togtema ◽  
Ingeborg Zehbe
Keyword(s):  
Human Papillomavirus ◽  
High Risk ◽  
Asian American ◽  
Protein Interactions ◽  
Host Protein ◽  
Cellular Proteins ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Host Proteins ◽  
E6 Protein

AbstractHigh-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers with type 16 being the most frequent. Compared to the European Prototype (A1, denoted “EP”), the Asian-American (D2/D3, denoted “AA”) sub-lineage or “variant” is reported to have increased abilities to promote carcinogenesis. Few global interactome studies have looked at protein-protein interactions (PPIs) between host proteins and variants of the key transforming E6 protein. We applied a primary human foreskin keratinocyte model transduced with EP and AA variant E6 genes and co-immunoprecipitated expressed E6 proteins along with interacting cellular proteins to detect virus-host binding partners. We reasoned that, due to single nucleotide polymorphisms, AAE6 and EPE6 may have unique PPIs with host cellular proteins—conferring gain or loss of function—resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to each of AAE6 and EPE6, along with 13 E6 targets common to both AAE6 and EPE6. We also applied a more inclusive process based on pathway selection and discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets between the two sub-lineages. Interpretations for both approaches were made using databases such as UniProt, BioGRID and Reactome. Detected E6 targets are implicated in important hallmarks of cancer: deregulating Notch and other signaling, energetics and hypoxia, DNA replication and repair, and immune response. Validation experiments, such as reverse co-immunoprecipitation and RNA interference, are required to substantiate these findings. Here, we provide an unprecedented resource for new research questions in HR HPV biology. The current data also underline our lab’s driving hypothesis that E6, being a “master regulator” in HPV-positive cancers, is an excellent candidate for anti-cancer treatment strategies.Author SummaryChronic infection with high-risk human papillomavirus (HPV) type 16 is the most prevalent cause of cervical and other mucosal cancers. The E6 oncoproteins of the European Prototype (EP) and the Asian-American (AA) HPV variants differentially promote carcinogenesis. We looked at protein-protein interactions between host proteins and two key HPV variant E6 proteins of these strains to reveal how high risk HPVs cause cancer, based on the proteins they bind to in infected cells. Our methodology combined molecular biology and data mining techniques using widely available databases. We confirmed and discovered novel virus-host associations that explained how HPV AA and EP variants differ in their carcinogenic capabilities, and confirmed the candidacy of the E6 protein as a viable target for HPV therapies.

scholarly journals Temporal Viral Genome-Protein Interactions Define Distinct Stages of Productive Herpesviral Infection

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Jill A. Dembowski ◽  
Neal A. DeLuca
Keyword(s):  
Life Cycle ◽  
Protein Interactions ◽  
Viral Genome ◽  
Isotope Labeling ◽  
De Novo ◽  
Affinity Purification ◽  
Human Pathogens ◽  
Host Proteins ◽  
Viral Dna ◽  
Hsv 1

ABSTRACTHerpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host. To investigate dynamic interactions between herpes simplex virus type 1 (HSV-1) DNA and viral and host proteins throughout infection, we developed an approach to identify proteins that associate with the infecting viral genome from nuclear entry through packaging. To accomplish this, virus stocks were prepared in the presence of ethynyl-modified nucleotides to enable covalent tagging of viral genomes after infection for analysis of viral genome-protein interactions by imaging or affinity purification. Affinity purification was combined with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry to enable the distinction between proteins that were brought into the cell by the virus or expressed within the infected cell before or during infection. We found that input viral DNA progressed within 6 h through four temporal stages where the genomes sequentially (i) interacted with intrinsic antiviral and DNA damage response proteins, (ii) underwent a robust transcriptional switch mediated largely by ICP4, (iii) engaged in replication, repair, and continued transcription, and then (iv) transitioned to a more transcriptionally inert state engagingde novo-synthesized viral structural components while maintaining interactions with replication proteins. Using a combination of genetic, imaging, and proteomic approaches, we provide a new and temporally compressed view of the HSV-1 life cycle based on input genome-proteome dynamics.IMPORTANCEHerpesviruses are highly prevalent and ubiquitous human pathogens. Studies of herpesviruses and other viruses have previously been limited by the ability to directly study events that occur on the viral DNA throughout infection. We present a new powerful approach, which allows for the temporal investigation of viral genome-protein interactions at all phases of infection. This work has integrated many results from previous studies with the discovery of novel factors potentially involved in viral infection that may represent new antiviral targets. In addition, the study provides a new view of the HSV-1 life cycle based on genome-proteome dynamics.

scholarly journals Virus–Host Protein–Protein Interactions between Human Papillomavirus 16 E6 A1 and D2/D3 Sub-Lineages: Variances and Similarities

2020 ◽  
Vol 21 (21) ◽  
pp. 7980
Author(s):  
Guillem Dayer ◽  
Mehran L. Masoom ◽  
Melissa Togtema ◽  
Ingeborg Zehbe
Keyword(s):  
Human Papillomavirus ◽  
Asian American ◽  
Protein Interactions ◽  
Host Protein ◽  
Cellular Proteins ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Human Papillomavirus 16 ◽  
Novel Approach ◽  
Causative Agents

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers, with type 16 being the most frequent. Compared to the European Prototype (EP; A1), the Asian-American (AA; D2/D3) sub-lineage seems to have increased abilities to promote carcinogenesis. Here, we studied protein–protein interactions (PPIs) between host proteins and sub-lineages of the key transforming E6 protein. We transduced human keratinocyte with EP or AA E6 genes and co-immunoprecipitated E6 proteins along with interacting cellular proteins to detect virus–host binding partners. AAE6 and EPE6 may have unique PPIs with host cellular proteins, conferring gain or loss of function and resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to AAE6 and EPE6, along with 13 E6 targets common to both. A novel approach based on pathway selection discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets. Interpretations were made using databases, such as UniProt, BioGRID, and Reactome. Detected E6 targets were differentially implicated in important hallmarks of cancer: deregulating Notch signaling, energetics and hypoxia, DNA replication and repair, and immune response.

scholarly journals Bromodomain Protein 4 Mediates the Papillomavirus E2 Transcriptional Activation Function

2006 ◽  
Vol 80 (9) ◽  
pp. 4276-4285 ◽  
Author(s):  
Michal-Ruth Schweiger ◽  
Jianxin You ◽  
Peter M. Howley
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Viral Genome ◽  
Regulatory Protein ◽  
Activation Function ◽  
Transactivation Domain ◽  
Genome Maintenance ◽  
Mitotic Chromosomes ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.

scholarly journals Proximity Biotin Labeling Reveals Kaposi’s Sarcoma-Associated Herpesvirus Interferon Regulatory Factor Networks

2021 ◽  
Vol 95 (9) ◽  
Author(s):  
Ashish Kumar ◽  
Michelle Salemi ◽  
Resham Bhullar ◽  
Sara Guevara-Plunkett ◽  
Yuanzhi Lyu ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Immune Regulation ◽  
Protein Complex ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Viral Proteins ◽  
Innate Immune ◽  
Host Protein ◽  
Cellular Proteins ◽  
Affinity Precipitation

ABSTRACT Studies on “hit-and-run” effects by viral proteins are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled identification of both static and dynamic protein-protein interactions. In this study, we applied a PL method by generating recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV, a gammaherpesvirus, uniquely encodes four interferon regulatory factors (IRF-1 to -4) that suppress host interferon responses, and we examined KSHV IRF-1 and IRF-4 neighbor proteins to identify cellular proteins involved in innate immune regulation. PL identified 213 and 70 proteins as neighboring proteins of viral IRF-1 (vIRF-1) and vIRF-4 during viral reactivation, and 47 proteins were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological roles such as mRNA processing and transcriptional regulation by TP53. Innate immune regulation by these commonly interacting 44 cellular proteins was examined with small interfering RNAs (siRNAs), and the splicing factor 3B family proteins were found to be associated with interferon transcription and to act as suppressors of KSHV reactivation. We propose that recombinant mini-TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication and that selective splicing factors have a function in the regulation of innate immune responses. IMPORTANCE Viral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to the use of affinity precipitation approaches, the majority of studies have focused on how the virus takes advantage of the newly formed protein interactions for its own replication. Proximity labeling (PL), however, can also highlight transient and negative effects—those interactions which lead to dissociation from the existing protein complex. Here, we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

scholarly journals The HPV16 E2 transcriptional regulator mode of action depends on the physical state of the viral genome.

2005 ◽  
Vol 52 (4) ◽  
pp. 823-832 ◽  
Author(s):  
Marcin T Schmidt ◽  
Agnieszka K Olejnik ◽  
Anna Goździcka-Józefiak
Keyword(s):  
Control Region ◽  
Viral Genome ◽  
Hormone Receptors ◽  
Long Control Region ◽  
Viral Dna ◽  
E2 Protein ◽  
E6 And E7 Oncoproteins ◽  
E6 And E7 ◽  
Episomal Dna

Human papillomavirus (HPV) infection is a major risk factor for the development of cervical cancer. The HPV-induced immortalization of epithelial cell usually requires integration of the viral DNA into the host cell genome. The integration event causes disruption of the E2 gene and this is followed by overexpression of the E6 and E7 oncoproteins. The E2 protein is a transcription factor that regulates expression of the E6 and E7 oncoproteins by binding to four sites within the viral long control region. We used an in vitro cell culture model to explore the role of the E2 protein in the transcriptional control of the HPV16 long control region. Employing transient and stable transfection experiments we simulated the episomal and integrated states of the viral genome, respectively. We show that the E2 protein up-regulates E6/E7 transcription from episomal DNA but represses it in the case of integrated DNA. The activator function of the E2 protein seems to counteract the repressive chromatin structure formed over episomal DNA. Steroid hormones and retinol also modulate oncogene transcription differently depending on the physical structure of the viral DNA. Our data suggest regulatory mechanisms involving interactions between the E2 protein and nuclear hormone receptors.

scholarly journals Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance

2010 ◽  
Vol 84 (22) ◽  
pp. 11747-11760 ◽  
Author(s):  
Amélie Fradet-Turcotte ◽  
Cary Moody ◽  
Laimonis A. Laimins ◽  
Jacques Archambault
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Nuclear Export ◽  
Viral Genome ◽  
Cellular Proliferation ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Genome Maintenance ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.

scholarly journals Biologi Molekuler Human Papilloma Virus

2018 ◽  
Vol 11 (1) ◽  
pp. 1
Author(s):  
Maya Savira
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Human Papilloma Virus ◽  
Cancer Cells ◽  
Persistent Infection ◽  
Malignant Disease ◽  
Risk Group ◽  
High Risk Group ◽  
Papilloma Virus ◽  
Carcinogenic Process

Persistent infection of the high-risk group Human Papilloma Virus (hrHPV) has been known to cause cervical cancer.Currently cervical cancer still ranks as the number two of most malignant disease in women. The infection of HPVthat lead to cancer cells to form can be known through the structure of the HPV virus itself and what particles in thevirus play a role in initiating the carcinogenic process of its host cell.

scholarly journals Actin-Modulating Protein Cofilin Is Involved in the Formation of Measles Virus Ribonucleoprotein Complex at the Perinuclear Region

2015 ◽  
Vol 89 (20) ◽  
pp. 10524-10531 ◽  
Author(s):  
Ritsuko Koga ◽  
Yukihiko Sugita ◽  
Takeshi Noda ◽  
Yusuke Yanagi ◽  
Shinji Ohno
Keyword(s):  
Viral Genome ◽  
Measles Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Host Protein ◽  
Cellular Proteins ◽  
N Protein ◽  
Phosphorylated Form ◽  
Rnp Complex ◽  
Infected Cells

ABSTRACTIn measles virus (MV)-infected cells, the ribonucleoprotein (RNP) complex, comprised of the viral genome and the nucleocapsid (N) protein, phosphoprotein (P protein), and large protein, assembles at the perinuclear region and synthesizes viral RNAs. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, interacts with the MV N protein and aids in the formation of the RNP complex. Knockdown of cofilin using the short hairpin RNA reduces the formation of the RNP complex after MV infection and that of the RNP complex-like structure after plasmid-mediated expression of MV N and P proteins. A lower level of formation of the RNP complex results in the reduction of viral RNA synthesis. Cofilin phosphorylation on the serine residue at position 3, an enzymatically inactive form, is increased after MV infection and the phosphorylated form of cofilin is preferentially included in the complex. These results indicate that cofilin plays an important role in MV replication by increasing formation of the RNP complex and viral RNA synthesis.IMPORTANCEMany RNA viruses induce within infected cells the structure called the ribonucleoprotein (RNP) complex in which viral RNA synthesis occurs. It is comprised of the viral genome and proteins that include the viral RNA polymerase. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, binds to the measles virus (MV) nucleocapsid protein and plays an important role in the formation of the MV RNP complex and MV RNA synthesis. The level of the phosphorylated form of cofilin, enzymatically inactive, is increased after MV infection, and the phosphorylated form is preferentially associated with the RNP complex. Our findings determined with cofilin will help us better understand the mechanism by which the RNP complex is formed in virus-infected cells and develop new antiviral drugs targeting the RNP complex.

scholarly journals Proximity Biotin Labeling Reveals KSHV Interferon Regulatory Factor Networks

2020 ◽  
Author(s):  
Ashish Kumar ◽  
Michelle Salemi ◽  
Resham Bhullar ◽  
Sara Guevara-Plunkett ◽  
Yuanzhi Lyu ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complex ◽  
Viral Protein ◽  
De Novo ◽  
Affinity Purification ◽  
Innate Immune ◽  
Host Protein ◽  
Cellular Proteins ◽  
Interacting Proteins ◽  
Affinity Precipitation

AbstractStudies on “HIT&RUN” effects by viral protein are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled study of both static and dynamic protein-protein interactions. Here we applied PL method with recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) in the genome, and we identified KSHV vIRF-1 and vIRF-4 interacting proteins during reactivation. Fusion of mini-TurboID with vIRF-1 or vIRF-4 did not interfere with KSHV gene expression, DNA replication, or de novo infections. PL identified 213 and 70 proteins for vIRF-1 and vIRF-4 respectively, which possibly interact during KSHV reactivation, and 47 of those were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological functions such as mRNA processing and transcriptional regulation by TP53. Involvement of commonly interacting 44 cellular proteins in innate immune regulation were examined by siRNAs, and we identified that splicing factor 3B (SF3B) family proteins were clearly involved in interferons transcription and suppressed KSHV reactivation. We propose that recombinant TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors may have a function beyond connecting two exon sequences to regulate innate immune responses.ImportanceViral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to use of affinity-precipitation approaches, the majority of our studies focused on how the virus takes advantage of the newly-formed protein interactions for its own replication. Proximity labeling (PL) however, can also highlight the transient and negative effects – those interactions which lead to dissociation from the existing protein complex. Here we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

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