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 An adenovirus type 5 E1A protein with a single amino acid substitution blocks wild-type E1A transactivation.

1987 ◽  
Vol 7 (3) ◽  
pp. 1004-1011 ◽  
Author(s):  
G M Glenn ◽  
R P Ricciardi
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Adenovirus Type ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Wild Type ◽  
Missense Mutant ◽  
E1a Gene ◽  
Adenovirus Type 5 ◽  
E1a Protein

The E1A gene of adenovirus type 5 encodes a 289-amino-acid (289R) protein that transactivates early adenovirus promoters. We showed that the 289R protein of the E1A missense mutant gene hr5 is novel in that it inhibits the wild-type (wt) E1A protein from stimulating transcription from each of the early viral promoters E2, E3, and E4. Since both the hr5 and wt genes produced similar levels of E1A proteins, the ability of hr5 E1A to block transactivation was attributed to the replacement of serine by asparagine as position 185. We confirmed that this single amino acid substitution was responsible for blocking transactivation by showing equal inhibition with an hr5-wt hybrid E1A gene containing this missense mutation as the only alteration. The smaller 243R E1A protein of hr5 was not necessary for inhibition. Transcriptional activity from each early promoter was inhibited by at least 50% when the hr5 and wt E1A genes were present in equimolar amounts; complete inhibition occurred with a fivefold molar excess of the hr5 gene. Two other E1A missense mutant genes (hr3 and hr4) with amino acid substitutions in close proximity to that of hr5 failed to block wt E1A-induced transcription when similarly tested. Also, the hr5 E1A gene failed to impede the pseudorabies immediate early gene from transactivating the adenovirus E3 promoter, demonstrating that hr5 E1A inhibits wt E1A activation at the transcriptional, rather than the posttranscriptional, level. Although several possibilities were considered to account for this inhibition, the most likely is that the nonfunctional hr5 E1A protein competes with the wt 289R protein for a cellular transcription factor required for transactivation.

An adenovirus type 5 E1A protein with a single amino acid substitution blocks wild-type E1A transactivation

1987 ◽  
Vol 7 (3) ◽  
pp. 1004-1011
Author(s):  
G M Glenn ◽  
R P Ricciardi
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Adenovirus Type ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Wild Type ◽  
Missense Mutant ◽  
E1a Gene ◽  
Adenovirus Type 5 ◽  
E1a Protein

The E1A gene of adenovirus type 5 encodes a 289-amino-acid (289R) protein that transactivates early adenovirus promoters. We showed that the 289R protein of the E1A missense mutant gene hr5 is novel in that it inhibits the wild-type (wt) E1A protein from stimulating transcription from each of the early viral promoters E2, E3, and E4. Since both the hr5 and wt genes produced similar levels of E1A proteins, the ability of hr5 E1A to block transactivation was attributed to the replacement of serine by asparagine as position 185. We confirmed that this single amino acid substitution was responsible for blocking transactivation by showing equal inhibition with an hr5-wt hybrid E1A gene containing this missense mutation as the only alteration. The smaller 243R E1A protein of hr5 was not necessary for inhibition. Transcriptional activity from each early promoter was inhibited by at least 50% when the hr5 and wt E1A genes were present in equimolar amounts; complete inhibition occurred with a fivefold molar excess of the hr5 gene. Two other E1A missense mutant genes (hr3 and hr4) with amino acid substitutions in close proximity to that of hr5 failed to block wt E1A-induced transcription when similarly tested. Also, the hr5 E1A gene failed to impede the pseudorabies immediate early gene from transactivating the adenovirus E3 promoter, demonstrating that hr5 E1A inhibits wt E1A activation at the transcriptional, rather than the posttranscriptional, level. Although several possibilities were considered to account for this inhibition, the most likely is that the nonfunctional hr5 E1A protein competes with the wt 289R protein for a cellular transcription factor required for transactivation.

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 A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation

2016 ◽  
Vol 198 (9) ◽  
pp. 1451-1463 ◽  
Author(s):  
Kelly A. Flanagan ◽  
Joseph D. Comber ◽  
Elizabeth Mearls ◽  
Colleen Fenton ◽  
Anna F. Wang Erickson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Sigma Factor ◽  
Late Gene ◽  
Late Gene Expression ◽  
Gene Encoding ◽  
Content Type ◽  
Regulatory Circuit ◽  
Gene Regulatory

ABSTRACTSpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell duringBacillus subtilissporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor σG. Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of σG. ThecsfBgene is ordinarily transcribed in the forespore only by the early-acting sigma factor σF. However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28,csfBwas also aberrantly transcribed later by σG, the very target of CsfB inhibition. This regulation ofcsfBby SpoIIQ Tyr-28 is specific, given that the expression of other σF-dependent genes was unaffected. Moreover, we identified a conserved element within thecsfBpromoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes σGactivity in the forespore as a channel component but also specifically maximizes σGactivity as part of a gene regulatory circuit that represses σG-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times.IMPORTANCECellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developingBacillus subtilisspore is directed by the alternative sigma factor σG. The activity of σGrequires a channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression. Here we report that the channel protein SpoIIQ also specifically maximizes σGactivity as part of a previously unknown regulatory circuit that prevents σGfrom activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB. The discovery of this regulatory circuit significantly expands our understanding of the gene regulatory network controlling late gene expression in the developingB. subtilisspore.

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 A Single Amino Acid Residue Change in the P Protein of Parainfluenza Virus 5 Elevates Viral Gene Expression

2008 ◽  
Vol 82 (18) ◽  
pp. 9123-9133 ◽  
Author(s):  
Khalid A. Timani ◽  
Dengyun Sun ◽  
Minghao Sun ◽  
Celia Keim ◽  
Yuan Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Viral Gene Expression ◽  
Viral Rna ◽  
Single Amino Acid ◽  
Viral Gene ◽  
Wild Type ◽  
V Protein ◽  
P Protein

ABSTRACT Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI−) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI− are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi−) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi−) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI− virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi−) is sufficient for elevated viral gene expression. Using mass spectrometry and 33P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication.

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.

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