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

2008 ◽  
Vol 190 (10) ◽  
pp. 3538-3545 ◽  
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.

scholarly journals In Vivo and In Vitro Analysis of Baculovirusie-2 Mutants

1999 ◽  
Vol 73 (3) ◽  
pp. 2460-2468 ◽  
Author(s):  
Elena A. Prikhod’ko ◽  
Albert Lu ◽  
Joyce A. Wilson ◽  
Lois K. Miller
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Late Gene ◽  
Wild Type ◽  
Late Gene Expression ◽  
Specific Effects ◽  
Viral Promoters ◽  
Occlusion Bodies ◽  
A Cell ◽  
Budded Virus

ABSTRACT Upon transient expression in cell culture, the ie-2gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: transactivation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived fromSpodoptera frugiperda but not in a cell line derived fromTrichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. Theie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form ofie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus,ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.

scholarly journals Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification

2005 ◽  
Vol 79 (8) ◽  
pp. 4918-4926 ◽  
Author(s):  
Kathryn M. Spink ◽  
Laimonis A. Laimins
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Viral Genome ◽  
Copy Number ◽  
Dna Amplification ◽  
Regulatory Elements ◽  
Late Gene ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Viral Copy Number

ABSTRACT The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine β-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.

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

2002 ◽  
Vol 83 (8) ◽  
pp. 2015-2023 ◽  
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.

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

2001 ◽  
Vol 75 (20) ◽  
pp. 10005-10013 ◽  
Author(s):  
Loren del Mar Peña ◽  
Laimonis A. Laimins
Keyword(s):  
Gene Expression ◽  
Dnase I ◽  
Human Papillomaviruses ◽  
Open Reading Frame ◽  
Late Gene ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Undifferentiated Cells ◽  
Late Transcript ◽  
Chromatin Rearrangement

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.

scholarly journals Papillomavirus Capsid Protein Expression Level Depends on the Match between Codon Usage and tRNA Availability

1999 ◽  
Vol 73 (6) ◽  
pp. 4972-4982 ◽  
Author(s):  
Jian Zhou ◽  
Wen Jun Liu ◽  
Shi Wen Peng ◽  
Xiao Yi Sun ◽  
Ian Frazer
Keyword(s):  
Gene Expression ◽  
Codon Usage ◽  
Fluorescent Protein ◽  
In Vitro Translation ◽  
Late Gene ◽  
Wild Type ◽  
Late Gene Expression ◽  
Late Genes ◽  
Codon Composition ◽  
L1 And L2

ABSTRACT Translation of mRNA encoding the L1 and L2 capsid proteins of papillomavirus (PV) is restricted in vivo to differentiated epithelial cells, although transcription of the L1 and L2 late genes occurs more widely. The codon composition of PV late genes is quite different from that of most mammalian genes. To test the possibility that PV late gene codon composition determines the efficiency of PV late gene expression in some cell types, synthetic bovine papillomavirus type 1 (BPV1) late genes were constructed with codon composition modified to resemble the typical mammalian gene. Expression of these genes from a strong promoter in Cos-1 cells was compared with expression of wild-type BPV1 late genes from the same promoter. Both unmodified and modified PV late genes were transcribed in Cos-1 cells, but only the codon-modified genes were translated. In vitro translation of wild-type but not synthetic BPV1 L1 mRNA was markedly enhanced by addition of aminoacyl-tRNAs. Codon composition thus limits BPV1 late gene translation in Cos-1 cells, and this limitation can be overcome by modification of the codon composition of the genes or by provision of excess tRNA. Replacement of codons in the green fluorescent protein (gfp) gene with those frequently used in PV late genes did not alter gfp transcription in Cos-1 cells but almost abolished translation, supporting the hypothesis that the observed differences in efficiency of translation of modified and unmodified PV capsid genes were related to codon usage rather than mRNA structure. As tRNA populations vary within and between tissues in the same eukaryotic organism, we speculate that matching of tRNA availability to codon usage may be one determinant of the restriction of expression of PV late genes to differentiated epithelium.

scholarly journals Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

2001 ◽  
Vol 75 (9) ◽  
pp. 4297-4307 ◽  
Author(s):  
Yuqiao Shen ◽  
Galila Kitzes ◽  
Julie A. Nye ◽  
Ali Fattaey ◽  
Terry Hermiston
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Human Cancer ◽  
Adenovirus Type ◽  
Single Amino Acid ◽  
Late Gene ◽  
Wild Type ◽  
Late Gene Expression ◽  
Human Cancer Cells ◽  
Adenovirus Type 5

ABSTRACT The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.

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

2015 ◽  
Vol 90 (1) ◽  
pp. 599-604 ◽  
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.

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

2007 ◽  
Vol 81 (12) ◽  
pp. 6761-6764 ◽  
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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