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 The [KIL-d] Element Specifically Regulates Viral Gene Expression in Yeast

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 601-609 ◽  
Author(s):  
Zsolt Tallóczy ◽  
Rebecca Mazar ◽  
Denise E Georgopoulos ◽  
Fausto Ramos ◽  
Michael J Leibowitz
Keyword(s):  
Gene Expression ◽  
Phenotypic Expression ◽  
Viral Gene Expression ◽  
Virus Genome ◽  
Viral Rna ◽  
High Rate ◽  
Viral Gene ◽  
Double Stranded Rna ◽  
Yeast Prions ◽  
Killer Virus

Abstract The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are “healed” in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10−4–10−5 and reappears with variegated phenotypic expression with a frequency of ≥10−3. This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

scholarly journals Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

2014 ◽  
Vol 89 (1) ◽  
pp. 195-207 ◽  
Author(s):  
Bishi Fu ◽  
Ersheng Kuang ◽  
Wenwei Li ◽  
Denis Avey ◽  
Xiaojuan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Artificial Chromosome ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Artificial Chromosome ◽  
Lytic Replication ◽  
Viral Gene ◽  
Wild Type ◽  
Lytic Reactivation ◽  
Infected Cells

ABSTRACTWe have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838–1850, 2008,http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.IMPORTANCEKSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

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.

Single Amino Acid Residue in the Extracellular Portion of Transmembrane Segment 2 in the Nicotinic α7 Acetylcholine Receptor Modulates Sensitivity to Ketamine

2004 ◽  
Vol 100 (3) ◽  
pp. 657-662 ◽  
Author(s):  
Kenny K. Ho ◽  
Pamela Flood
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Nicotinic Acetylcholine Receptors ◽  
Nicotinic Receptor ◽  
Acetylcholine Receptors ◽  
Nicotinic Receptors ◽  
Single Amino Acid ◽  
Wild Type ◽  
Alpha7 Nicotinic Receptor ◽  
Homologous Amino Acid

Background Ketamine inhibits the activation of both heteromeric and homomeric nicotinic acetylcholine receptors. The site of molecular interaction is unknown. Methods The inhibition of alpha7 nicotinic acetylcholine receptors by ketamine was compared to that of 5-hydroxytryptamine-3A (5HT3A) receptors that are resistant to ketamine inhibition in Xenopus laevis oocytes. To determine whether the region of transmembrane segments 2 and 3 is relevant for ketamine inhibition of nicotinic receptors, the authors identified single amino acid residues that differ in the sequence alignment of the two proteins. They created 22 mutant alpha7 nicotinic receptors that contain the single homologous amino acid residue in the 5HT3A sequence. Results Of the 22 mutant alpha7 nicotinic receptors tested, only one (alpha7 A258S) was significantly resistant to 20 microM ketamine. The ketamine concentration response relationship for the alpha7 A258S mutant was shifted to the right with the IC50 for ketamine increased from 17 +/- 2 for wild type to 30 +/- 3 microM in the mutant (P < 0.001). Agonist activation was unchanged by the mutation. The homologous amino acid residue in the 5HT3A receptor was mutated to the alanine that occurs in the wild-type nicotinic receptor. This mutation made the previously insensitive 5HT3A receptor sensitive to ketamine (P < 0.001). Conclusions Conservative mutation of a single amino acid in the extracellular transmembrane segment 2 domain induces resistance to ketamine inhibition in the alpha7 nicotinic receptor and sensitivity to inhibition in the 5HT3A receptor. This region may represent a ketamine binding site in the alpha7 nicotinic receptor, or it may be an important transduction site for ketamine action.

scholarly journals Measles Viruses Possessing the Polymerase Protein Genes of the Edmonston Vaccine Strain Exhibit Attenuated Gene Expression and Growth in Cultured Cells and SLAM Knock-In Mice

2008 ◽  
Vol 82 (23) ◽  
pp. 11979-11984 ◽  
Author(s):  
Makoto Takeda ◽  
Shinji Ohno ◽  
Maino Tahara ◽  
Hiroki Takeuchi ◽  
Yuta Shirogane ◽  
...  
Keyword(s):  
Gene Expression ◽  
Measles Virus ◽  
Cultured Cells ◽  
Lymphocyte Activation ◽  
Viral Gene Expression ◽  
Promoter Sequence ◽  
Virus Genome ◽  
Viral Gene ◽  
Wild Type ◽  
Vaccine Type

ABSTRACT Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.

scholarly journals Residues 231 to 280 of the Epstein-Barr Virus Nuclear Protein 2 Are Not Essential for Primary B-Lymphocyte Growth Transformation

1998 ◽  
Vol 72 (12) ◽  
pp. 9948-9954 ◽  
Author(s):  
Shizuko Harada ◽  
Ramana Yalamanchili ◽  
Elliott Kieff
Keyword(s):  
Gene Expression ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Nuclear Protein ◽  
Viral Gene Expression ◽  
Transient Transfection ◽  
Viral Gene ◽  
Wild Type ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is a transcriptional transactivator of cellular and viral gene expression and is essential for the transformation of resting human B lymphocytes into long-term lymphoblastoid cell lines (LCLs). Previous molecular genetic analyses identified three domains that are critical for transformation and showed that the rest of EBNA-2 is not critical. We now find that codons 231 to 280 that were part of one of the critical domains (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545–2554, 1991) can be deleted with only a small effect on the ability of EBNA-2 to transactivate gene expression. In transient transfection assays, EBNA-2 deleted for codons 231 to 280 accumulated to higher levels and was similar to wild-type EBNA-2 in activation of the BamC promoter and in association with RBPJk, a cellular transcription factor that is important for EBNA-2 interaction with promoter regulatory elements. However, EBNA-2 d231–280 activated the viral latent membrane protein 1 (LMP1) promoter with only 60% of wild-type efficiency. Recombinant EBVs specifically deleted for EBNA-2 codons 231 to 280 were efficient in initiating the transformation of resting primary human B lymphocytes into LCLs. However, these LCLs grew less well than wild-type EBV-transformed LCLs, and 4- to 10-fold more cells were required for outgrowth following limit dilution. EBNA-2 d231–280 accumulated to unusually high levels in the recombinant transformed LCLs, and this was associated with somewhat higher EBNA-1 and lower LMP1 expression, consistent with the near-wild-type activation of the BamC EBNA promoter and the abnormally low activation of the LMP1 promoter in transient transfection assays. Thus, EBNA-2 d231–280 modestly perturbed the regulation of viral gene expression and resulted in less LMP1, while having surprisingly subtle effects on LCL outgrowth. Deletion of EBNA-2 codons 292 to 310, which are closer to the site that specifies interaction with RBPJk, was more disruptive of RBPJk association and of the ability to transform B lymphocytes.

scholarly journals Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication

1998 ◽  
Vol 72 (8) ◽  
pp. 6629-6636 ◽  
Author(s):  
Chen Liang ◽  
Liwei Rong ◽  
Michael Laughrea ◽  
Lawrence Kleiman ◽  
Mark A. Wainberg
Keyword(s):  
Gene Expression ◽  
Point Mutations ◽  
Viral Gene Expression ◽  
Initiation Site ◽  
Viral Rna ◽  
Viral Gene ◽  
Rna Packaging ◽  
Immunodeficiency Virus ◽  
Dimerization Initiation Site

ABSTRACT The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3′ end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.

scholarly journals The Late Promoter of the Human Cytomegalovirus Viral DNA Polymerase Processivity Factor Has an Impact on Delayed Early and Late Viral Gene Products but Not on Viral DNA Synthesis

2007 ◽  
Vol 81 (12) ◽  
pp. 6197-6206 ◽  
Author(s):  
Hiroki Isomura ◽  
Mark F. Stinski ◽  
Ayumi Kudoh ◽  
Sanae Nakayama ◽  
Satoko Iwahori ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Dna ◽  
Recombinant Viruses ◽  
Tata Element ◽  
Processivity Factor

ABSTRACT Transcription of the DNA polymerase processivity factor gene (UL44) of human cytomegalovirus initiates at three distinct start sites, which are differentially regulated during productive infection. Two of these start sites, the distal and proximal sites, are active at early times, and the middle start site is active at only late times after infection (F. Leach and E. S. Mocarski, J. Virol. 63:1783-1791, 1989). Compared to the wild type, UL44 gene expression was lower for recombinant viruses with the distal or the middle TATA element mutated. The transcripts initiating from the distal or middle start site facilitated late viral gene expression. The level of viral DNA synthesis was affected by mutation of the distal TATA element. In contrast, mutation of the middle TATA element did not affect the level of viral DNA synthesis, but it did affect significantly the level of late viral gene expression. Recombinant viruses with the distal or middle TATA element mutated grew more slowly than the wild type at both low and high multiplicities of infection. Reduced expression of the UL44 gene from the late middle viral promoter correlated with decreased late viral protein expression and decreased viral growth.

scholarly journals Regulation of measles virus gene expression by P protein coiled-coil properties

2019 ◽  
Vol 5 (5) ◽  
pp. eaaw3702 ◽  
Author(s):  
Louis-Marie Bloyet ◽  
Antoine Schramm ◽  
Carine Lazert ◽  
Bertrand Raynal ◽  
Maggy Hologne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Measles Virus ◽  
Narrow Range ◽  
Rna Viruses ◽  
Coiled Coil ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Large Protein ◽  
P Protein ◽  
Hydrophobic Anchor

The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the “a” or “d” hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness.

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