scholarly journals Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells

2003 ◽  
Vol 77 (1) ◽  
pp. 535-545 ◽  
Author(s):  
Liqun Lu ◽  
Quansheng Du ◽  
Nor Chejanovsky
Keyword(s):  
Steady State ◽  
Spodoptera Littoralis ◽  
Autographa Californica ◽  
Early Gene ◽  
Immediate Early ◽  
Cosmid Library ◽  
Functional Protein ◽  
Early Protein ◽  
Multiple Nucleopolyhedrovirus ◽  
Steady State Levels

ABSTRACT Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles

2006 ◽  
Vol 80 (12) ◽  
pp. 5886-5896 ◽  
Author(s):  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Steady State ◽  
Human Cytomegalovirus ◽  
Virus Titer ◽  
State Level ◽  
Early Gene ◽  
Tegument Protein ◽  
Cyclin Dependent Kinase ◽  
Viral Dna ◽  
Infected Cells ◽  
Steady State Levels

ABSTRACT We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

scholarly journals Recruitment of Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 to the Virus Particle

2010 ◽  
Vol 84 (9) ◽  
pp. 4682-4696 ◽  
Author(s):  
Kevin Maringer ◽  
Gillian Elliott
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Early Gene ◽  
Immediate Early ◽  
Independent Manner ◽  
Early Protein ◽  
Simplex Virus

ABSTRACT Although the herpes simplex virus type 1 (HSV-1) tegument is comprised of a large number of viral and cellular proteins, how and where in the cell these proteins are recruited into the virus structure is poorly understood. We have shown previously that the immediate-early gene product ICP0 is packaged by a mechanism dependent on the major tegument protein VP22, while others have shown a requirement for ICP27. We now extend our studies to show that ICP0 packaging correlates directly with the ability of ICP0 to complex with VP22 in infected cells. ICP27 is not, however, present in this VP22-ICP0 complex but is packaged into the virion in a VP22- and ICP0-independent manner. Biochemical fractionation of virions indicated that ICP0 associates tightly with the virus capsid, but intranuclear capsids contained no detectable ICP0. The RING finger domain of ICP0 and the N terminus of VP22 were both shown to be essential but not sufficient for ICP0 packaging and complex formation. Strikingly, however, the N-terminal region of VP22, while unable to form a complex with ICP0, inhibited its translocation from the nucleus to the cytoplasm. PML degradation by ICP0 was efficient in cells infected with this VP22 mutant virus, confirming that ICP0 retains activity. Hence, we would suggest that VP22 is an important molecular partner of ICP0 that controls at least one of its activities: its assembly into the virion. Moreover, we propose that the pathway by which VP22 recruits ICP0 to the virion may begin in the nucleus prior to ICP0 translocation to its final site of assembly in the cytoplasm.

scholarly journals Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

2014 ◽  
Vol 89 (1) ◽  
pp. 373-383 ◽  
Author(s):  
Xue Ying Tao ◽  
Jae Young Choi ◽  
Woo Jin Kim ◽  
Saes Byeol An ◽  
Qin Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Gene Knockout ◽  
Autographa Californica ◽  
Early Gene ◽  
Expression Vectors ◽  
Knockout Mutant ◽  
Transfected Cells ◽  
Multiple Nucleopolyhedrovirus ◽  
Baculovirus Infection

ABSTRACTORF11 (ac11) ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role ofac11in the baculovirus life cycle, anac11knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5′ rapid amplification of cDNA ends (RACE) analyses revealed thatac11is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion ofac11did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating thatac11is required for ODV envelopment. These results therefore demonstrate thatac11is an early gene that is essential for BV production and ODV envelopment.IMPORTANCEBaculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study,ac11was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

scholarly journals The Human Cytomegalovirus 86-Kilodalton Major Immediate-Early Protein Interacts Physically and Functionally with Histone Acetyltransferase P/CAF

2000 ◽  
Vol 74 (16) ◽  
pp. 7230-7237 ◽  
Author(s):  
L. A. Bryant ◽  
P. Mixon ◽  
M. Davidson ◽  
A. J. Bannister ◽  
T. Kouzarides ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Histone Acetyltransferase ◽  
Early Gene ◽  
Immediate Early ◽  
Productive Infection ◽  
Early Protein ◽  
Cellular Genes ◽  
Immediate Early Proteins ◽  
Target Promoters

ABSTRACT The major immediate-early proteins of human cytomegalovirus (HCMV) play a pivotal role in controlling viral and cellular gene expression during productive infection. As well as negatively autoregulating its own promoter, the HCMV 86-kDa major immediate early protein (IE86) activates viral early gene expression and is known to be a promiscuous transcriptional regulator of cellular genes. IE86 appears to act as a multimodal transcription factor. It is able to bind directly to target promoters to activate transcription but is also able to bridge between upstream binding factors such as CREB/ATF and the basal transcription complex as well as interacting directly with general transcription factors such as TATA-binding protein and TFIIB. We now show that IE86 is also able to interact directly with histone acetyltransferases during infection. At least one of these factors is the histone acetyltransferase CBP-associated factor (P/CAF). Furthermore, we show that this interaction results in synergistic transactivation by IE86 of IE86-responsive promoters. Recruitment of such chromatin-remodeling factors to target promoters by IE86 may help explain the ability of this viral protein to act as a promiscuous transactivator of cellular genes.

Integrin-dependent induction of early growth response genes in capillary endothelial cells

1996 ◽  
Vol 109 (12) ◽  
pp. 2855-2863 ◽  
Author(s):  
L.E. Dike ◽  
D.E. Ingber
Keyword(s):  
Extracellular Matrix ◽  
Steady State ◽  
Gene Activation ◽  
State Level ◽  
Gene Induction ◽  
Growth Cycle ◽  
Immediate Early ◽  
Capillary Endothelial Cells ◽  
Steady State Levels ◽  
Suspended Cells

Studies were carried out to explore how extracellular matrix molecules, such as fibronectin (FN), promote capillary endothelial (CE) cell growth. When G0-synchronized cells were plated on FN-coated dishes, expression of the immediate-early mRNAs, c-fos, c-myc and c-jun, was rapidly induced, even in the absence of serum or soluble growth factors. Moreover, plating cells on different FN densities (5-200 micrograms/150 mm dish), resulted in a dose-dependent increase in the steady state levels of these mRNAs. Addition of FGF potentiated gene activation and was required for maximal DNA synthesis, however, the overall steady-state level of gene induction was dictated primarily by the density of immobilized FN. Expression of junB also was induced when suspended cells bound RGD-peptide coated microbeads that promote integrin clustering, but not when the suspended cells bound beads coated with other receptor ligands (e.g. acetylated low density protein) or when they were stimulated by soluble FN or FGF in the absence of substrate adhesion. c-Jun exhibited a similar requirement for gene induction except that it also was partially induced by binding to soluble FN alone. In contrast, c-fos expression was induced by all stimuli tested. Interestingly, inhibition of Na+/H+ exchange using hexamethylene-amiloride prevented most of the FN-induced increase in c-jun expression whereas it was relatively ineffective when cells were simultaneously stimulated by both FN and FGF. These data demonstrate that cell adhesion to extracellular matrix and associated integrin binding can directly activate signaling cascades in quiescent CE cells that lead to induction of immediate-early genes associated with the G0/G1 transition and thereby, stimulate these cells to reenter the growth cycle.

scholarly journals E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection.

1992 ◽  
Vol 12 (10) ◽  
pp. 4364-4374 ◽  
Author(s):  
M Wade ◽  
T F Kowalik ◽  
M Mudryj ◽  
E S Huang ◽  
J C Azizkhan
Keyword(s):  
Human Cytomegalovirus ◽  
Dihydrofolate Reductase ◽  
Hcmv Infection ◽  
Cyclin A ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Gene Promoters ◽  
Cellular Gene ◽  
Early Protein

The adenovirus immediate-early protein E1A activates the adenovirus E2 promoter and several cellular gene promoters through transcription factor E2F. The immediate-early proteins of human cytomegalovirus (HCMV) can complement an E1A-deficient adenovirus mutant and activate the adenovirus E2 promoter. HCMV also has been shown to activate the adenovirus E2 promoter. On the basis of these findings, we have investigated whether HCMV can activate the promoter of the cellular dihydrofolate reductase (DHFR) gene, which requires E2F binding for maximal promoter activity. We show that HCMV activates the DHFR promoter and that products of the HCMV major immediate-early gene region mediate the activation of the promoter specifically through the E2F site. We used gel mobility shift assays to search for potential molecular mechanisms for this activation and found an "infection-specific" multimeric complex that bound to the E2F sites in the DHFR and E2 promoters in extracts from HCMV-infected cells but not in extracts from uninfected cells. Several antibodies against HCMV immediate-early gene products had no effect on this infection-specific complex. Subsequently, the complex was found to contain E2F, cyclin A, p33cdk2, and p107 and to be similar to S-phase-specific complexes that recently have been identified in several cell types. A functional role for the binding of the cyclin A-p33cdk2 complex to cellular gene promoters has yet to be demonstrated; however, HCMV infection causes the induction of both cellular DNA replication and transcription of growth-related genes containing E2F sites in their promoters. The findings described above therefore may relate to both of these effects of HCMV infection. We also provide evidence that some of the molecular events associated with adenovirus infection are different from those associated with HCMV infection.

scholarly journals Control of VP16 Translation by the Herpes Simplex Virus Type 1 Immediate-Early Protein ICP27

2005 ◽  
Vol 79 (7) ◽  
pp. 4120-4131 ◽  
Author(s):  
Kimberly S. Ellison ◽  
Robert A. Maranchuk ◽  
Kelly L. Mottet ◽  
James R. Smiley
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Export ◽  
Early Gene ◽  
Immediate Early ◽  
Regulatory Function ◽  
Early Protein ◽  
Simplex Virus ◽  
Nuclear Rna Export

ABSTRACT Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of gene expression that modulates the synthesis and maturation of viral and cellular mRNAs. Processes that are affected by ICP27 include transcription, pre-mRNA splicing, polyadenylation, and nuclear RNA export. We have examined how ICP27 influences the expression of the essential HSV tegument protein and transactivator of immediate-early gene expression VP16. We monitored the effects of ICP27 on the levels, nuclear export, and polyribosomal association of VP16 mRNA and on the amount and stability of VP16 protein. Deletion of ICP27 reduced the levels of VP16 mRNA without altering its nuclear export or the stability of the encoded protein. However, the translational yield of the VP16 mRNA produced in the absence of ICP27 was reduced 9- to 80-fold relative to that for wild-type infection, suggesting a defect in translation. In the absence of ICP27, the majority of cytoplasmic VP16 mRNA was not associated with actively translating polyribosomes but instead cosedimented with 40S ribosomal subunits, indicating that the translational defect is likely at the level of initiation. These effects were mRNA specific, as polyribosomal analysis of two cellular transcripts (glyceraldehyde-3-phosphate dehydrogenase and β-actin) and two early HSV transcripts (thymidine kinase and ICP8) indicated that ICP27 is not required for efficient translation of these mRNAs. Thus, we have uncovered a novel mRNA-specific translational regulatory function of ICP27.

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