Differentiation-Dependent Chromatin Rearrangement Coincides with Activation of Human Papillomavirus Type 31 Late Gene Expression

ABSTRACT The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation status of the host cell. While early genes are expressed during the initial stages of viral infection, late gene expression occurs in the suprabasal layers of the cervical epithelium. Late genes encode E1^E4, a cytosolic protein, and capsid proteins L1 and L2. We have mapped over 30 initiation sites for late transcripts and show that the transcripts initiate in a 200-nucleotide region within the E7 open reading frame. The mechanisms regulating the activation of late gene expression, however, are not yet understood. DNase I hypersensitivity analysis of HPV-31 chromatin in cell lines that maintain viral genomes extrachromosomally indicates that a major shift in nuclease digestion occurs upon differentiation. In undifferentiated cells, hypersensitive regions exist in the upstream regulatory region proximal to the E6 open reading frame. Upon differentiation, a region between nucleotides 659 and 811 in the E7 open reading frame becomes accessible to DNase I. These results indicate that the late transcript initiation region becomes accessible to transcription factor binding upon differentiation. Several complexes mediate chromatin rearrangement, and we tested whether histone acetylation was sufficient for late transcript activation. Treatment with the histone deacetylase inhibitor trichostatin A was found to be insufficient to activate late gene expression in undifferentiated cells. However, it did activate expression of early transcripts. These results suggest that chromatin remodeling around the late promoter occurs upon epithelial differentiation and that mechanisms in addition to histone deacetylation contribute to activation of late gene expression.

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Murine Gammaherpesvirus 68 Open Reading Frame 24 Is Required for Late Gene Expression after DNA Replication

Journal of Virology ◽

10.1128/jvi.02726-06 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6761-6764 ◽

Cited By ~ 34

Author(s):

Elaine Wong ◽

Ting-Ting Wu ◽

Nichole Reyes ◽

Hongyu Deng ◽

Ren Sun

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Viral Replication ◽

Open Reading Frame ◽

Late Gene ◽

Late Gene Expression ◽

Reading Frame ◽

Murine Gammaherpesvirus ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT Open reading frame 24 (ORF24) of murine gammaherpesvirus 68 (MHV-68) is conserved among beta- and gammaherpesviruses; however, its function in viral replication has not been defined. Using MHV-68 as a model, we have identified ORF24 as being essential for viral replication. An ORF24-null virus was generated and shown to be defective in late gene expression. Expression of early genes, as well as viral genome replication, was not affected. Furthermore, the defect in late gene expression was likely due to a deficiency in transcription. Thus, we have identified an MHV-68 protein, ORF24, that is essential for the expression of viral late proteins yet dispensable for viral DNA replication.

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Transactivation by the E2 Protein of Oncogenic Human Papillomavirus Type 31 Is Not Essential for Early and Late Viral Functions

Journal of Virology ◽

10.1128/jvi.72.10.8115-8123.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8115-8123 ◽

Cited By ~ 66

Author(s):

Frank Stubenrauch ◽

Angela M. E. Colbert ◽

Laimonis A. Laimins

Keyword(s):

Gene Expression ◽

Life Cycle ◽

Human Keratinocytes ◽

Viral Life Cycle ◽

Human Papillomaviruses ◽

Late Gene ◽

Late Gene Expression ◽

Positive Role ◽

E2 Protein ◽

Biochemical Analyses

ABSTRACT The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN6GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.

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Suppression of Stromal Interferon Signaling by Human Papillomavirus 16

Journal of Virology ◽

10.1128/jvi.00458-19 ◽

2019 ◽

Vol 93 (19) ◽

Author(s):

Gaurav Raikhy ◽

Brittany L. Woodby ◽

Matthew L. Scott ◽

Grace Shin ◽

Julia E. Myers ◽

...

Keyword(s):

Gene Expression ◽

Innate Immunity ◽

Human Papillomavirus ◽

Life Cycle ◽

Immune Evasion ◽

Human Papillomaviruses ◽

Keratinocyte Differentiation ◽

Late Gene ◽

Late Gene Expression ◽

Viral Oncogene

ABSTRACT Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs) and HFKs containing either wild-type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Interferon (IFN)-stimulated genes (ISGs) and extracellular matrix remodeling genes were suppressed, the most prominent pathways affected. STAT1, IFNAR1, IRF3, and IRF7 were knocked down in stromal fibroblasts using lentiviral short hairpin RNA (shRNA) transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated with increased late keratinocyte differentiation but not decreased IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation. IMPORTANCE The persistence of high-risk human papillomavirus (HPV) infections is the key risk factor for developing HPV-associated cancers. The ability of HPV to evade host immunity is a critical component of its ability to persist. The environment surrounding a tumor is increasingly understood to be critical in cancer development, including immune evasion. Our studies show that HPV can suppress the expression of immune-related genes in neighboring fibroblasts in a three-dimensional (3D) model of human epithelium. This finding is significant, because it indicates that HPV can control innate immunity not only in the infected cell but also in the microenvironment. In addition, the ability of HPV to regulate stromal gene expression depends in part on the viral oncogene E5, revealing a new function for this protein as an immune evasion factor.

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Characterization of late gene expression factors lef-9 and lef-8 from Bombyx mori nucleopolyhedrovirus

Journal of General Virology ◽

10.1099/0022-1317-83-8-2015 ◽

2002 ◽

Vol 83 (8) ◽

pp. 2015-2023 ◽

Cited By ~ 16

Author(s):

Asha Acharya ◽

Karumathil P. Gopinathan

Keyword(s):

Gene Expression ◽

Bombyx Mori ◽

Late Gene ◽

Late Gene Expression ◽

Reading Frame ◽

Early Transcription ◽

A Site ◽

Translational Termination

Late gene expression factors, LEF-4, LEF-8, LEF-9 and P47 constitute the primary components of the Autographa californica multinucleocapsid polyhedrovirus (AcMNPV)-encoded RNA polymerase, which initiates transcription from late and very late promoters. Here, characterization of lef-9 and lef-8, which encode their corresponding counterparts, from Bombyx mori NPV is reported. Transcription of lef-9 initiated at two independent sites: from a GCACT sequence located at −38 nt and a CTCTT sequence located at −50 nt, with respect to the +1 ATG of the open reading frame. The 3′ end of the transcript was mapped to a site 17 nt downstream of a canonical polyadenylation signal located 7 nt downstream of the first of the two tandem translational termination codons. Maximum synthesis of LEF-9 was seen from 36 h post-infection (p.i.). The transcription of lef-8 initiated early in infection from a GTGCAAT sequence that differed in the corresponding region from its AcMNPV counterpart (GCGCAGT), with consequent elimination of the consensus early transcription start site motif (underlined). Peak levels of lef-8 transcripts were attained by 24 h p.i. Immunocopurification analyses suggested that there was an association between LEF-8 and LEF-9 in vivo.

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Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression

Journal of Bacteriology ◽

10.1128/jb.01993-07 ◽

2008 ◽

Vol 190 (10) ◽

pp. 3538-3545 ◽

Cited By ~ 5

Author(s):

Nikolai V. Ravin ◽

Jérôme Rech ◽

David Lane

Keyword(s):

Gene Expression ◽

Late Gene ◽

Northern Hybridization ◽

Partition Functions ◽

Wild Type ◽

Late Promoter ◽

Late Gene Expression ◽

Reading Frame ◽

Partition System ◽

Plasmid Partition

ABSTRACT The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage λ) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to λ, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3 −-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3 + fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3 +-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

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Interaction between ORF24 and ORF34 in the Kaposi's Sarcoma-Associated Herpesvirus Late Gene Transcription Factor Complex Is Essential for Viral Late Gene Expression

Journal of Virology ◽

10.1128/jvi.02157-15 ◽

2015 ◽

Vol 90 (1) ◽

pp. 599-604 ◽

Cited By ~ 29

Author(s):

Zoe H. Davis ◽

Charles R. Hesser ◽

Jimin Park ◽

Britt A. Glaunsinger

Keyword(s):

Gene Expression ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Late Gene ◽

Late Gene Expression ◽

Reading Frame ◽

Interaction Interface ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Transcription Factor Complex

Transcription of herpesviral late genes is stimulated after the onset of viral DNA replication but otherwise restricted. Late gene expression in gammaherpesviruses requires the coordination of six early viral proteins, termed viral transactivation factors (vTFs). Here, we mapped the organization of this protein complex for Kaposi's sarcoma-associated herpesvirus. Disruption of this complex via point mutation of the interaction interface between the open reading frame 24 (ORF24) and ORF34 vTFs ablated both late gene expression and viral replication.

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SarA of Staphylococcus aureus Binds to the sarA Promoter To Regulate Gene Expression

Journal of Bacteriology ◽

10.1128/jb.01826-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 2239-2243 ◽

Cited By ~ 21

Author(s):

Ambrose L. Cheung ◽

Koren Nishina ◽

Adhar C. Manna

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Dnase I ◽

Open Reading Frame ◽

Regulate Gene Expression ◽

Reading Frame ◽

Dnase I Footprinting ◽

Gel Shift ◽

Regulate Gene

ABSTRACT The 375-bp sarA open reading frame is driven by three promoters, P1, P3, and P2. Using gel shift and DNase I footprinting assays, we found that SarA binds to two 26-bp sequences and one 31-bp sequence within the P1 and P3 promoters, respectively. Together with the results of transcription analyses, our data indicate that SarA binds to its own promoter to down-regulate sarA expression.

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Association of Kaposi's Sarcoma-Associated Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene Expression

Journal of Virology ◽

10.1128/jvi.00272-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 6148-6154 ◽

Cited By ~ 28

Author(s):

Kevin Brulois ◽

Lai-Yee Wong ◽

Hye-Ra Lee ◽

Priyanka Sivadas ◽

Armin Ensser ◽

...

Keyword(s):

Gene Expression ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Late Gene ◽

Late Gene Expression ◽

Reading Frame ◽

Amino Terminal ◽

Late Genes ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Transcription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes.

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Adenovirus Serotype 5 L4-22K and L4-33K Proteins Have Distinct Functions in Regulating Late Gene Expression

Journal of Virology ◽

10.1128/jvi.02455-08 ◽

2009 ◽

Vol 83 (7) ◽

pp. 3049-3058 ◽

Cited By ~ 23

Author(s):

Susan J. Morris ◽

Keith N. Leppard

Keyword(s):

Gene Expression ◽

Stop Codon ◽

Late Phase ◽

Transcription Unit ◽

Late Gene ◽

Small Contribution ◽

Late Gene Expression ◽

Reading Frame ◽

Early Protein ◽

Serotype 5

ABSTRACT Adenoviruses express up to 20 distinct mRNAs from five major late transcription unit (MLTU) regions, L1 to L5, by differential splicing and polyadenylation of the primary transcript. MLTU expression is regulated at transcriptional and posttranscriptional levels. The L4-33K protein acts as a splicing factor to upregulate several MLTU splice acceptor sites as the late phase progresses. The L4 region also expresses a 22K protein whose sequence is related to the sequence of L4-33K. L4-22K is shown here also to have an important role in regulating the pattern of MLTU gene expression. An adenovirus genome containing a stop codon in the L4-22K open reading frame expressed low levels of both structural and nonstructural late proteins compared to the wild-type (wt) adenovirus genome; a decrease in intermediate proteins, IVa2 and IX, was also observed. However, early protein synthesis and replication were unaffected by the absence of L4-22K. Intermediate and late protein expression was restored to wt levels by L4-22K expressed in trans but not by L4-33K. Increased MLTU promoter activity, resulting from stabilization of the transcriptional activator IVa2 by L4-22K, made a small contribution to this restoration of late gene expression. However, the principal effect of L4-22K was on the processing of MLTU RNA into specific cytoplasmic mRNA. L4-22K selectively increased expression of penton mRNA and protein, whereas splicing to create penton mRNA is known not to be increased by L4-33K. These results indicate that L4-22K plays a key role in the early-late switch in MLTU expression, additional to and distinct from the role of L4-33K.

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