scholarly journals Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport.

1985 ◽  
Vol 5 (10) ◽  
pp. 2552-2558 ◽  
Author(s):  
L E Babiss ◽  
H S Ginsberg ◽  
J E Darnell
Keyword(s):  
Mrna Translation ◽  
Mutant Virus ◽  
Viral Mrna ◽  
Mrna Synthesis ◽  
Mrna Transport ◽  
Normal Delivery ◽  
Single Function ◽  
Infected Cells ◽  
Steady State Levels ◽  
Adenovirus E1b

Late in adenovirus infection, large amounts of viral mRNA accumulate while cell mRNA transport and translation decrease. Viruses deleted in the E1B region of type 5 adenovirus do not produce the same outcome: (i) viral mRNA synthesis by the mutants is normal, delivery to the cytoplasm is 50 to 75% of normal, but steady-state levels of viral mRNA are decreased 10-fold; (ii) cell mRNA synthesis and transport continue normally in the mutant virus-infected cell; and (iii) translation of preexisting cell mRNA which is disrupted in wild-type infection remains normal in mutant-virus-infected cells. Thus E1B proteins are required for accumulation of virus mRNA and for induction of the failure of host cell mRNA transport and translation. If a single function is involved, by inference the transport and some aspect of translation of mRNAs could be linked.

Adenovirus E1B proteins are required for accumulation of late viral mRNA and for effects on cellular mRNA translation and transport

1985 ◽  
Vol 5 (10) ◽  
pp. 2552-2558 ◽  
Author(s):  
L E Babiss ◽  
H S Ginsberg ◽  
J E Darnell
Keyword(s):  
Mrna Translation ◽  
Mutant Virus ◽  
Viral Mrna ◽  
Mrna Synthesis ◽  
Mrna Transport ◽  
Normal Delivery ◽  
Single Function ◽  
Infected Cells ◽  
Steady State Levels ◽  
Adenovirus E1b

Late in adenovirus infection, large amounts of viral mRNA accumulate while cell mRNA transport and translation decrease. Viruses deleted in the E1B region of type 5 adenovirus do not produce the same outcome: (i) viral mRNA synthesis by the mutants is normal, delivery to the cytoplasm is 50 to 75% of normal, but steady-state levels of viral mRNA are decreased 10-fold; (ii) cell mRNA synthesis and transport continue normally in the mutant virus-infected cell; and (iii) translation of preexisting cell mRNA which is disrupted in wild-type infection remains normal in mutant-virus-infected cells. Thus E1B proteins are required for accumulation of virus mRNA and for induction of the failure of host cell mRNA transport and translation. If a single function is involved, by inference the transport and some aspect of translation of mRNAs could be linked.

scholarly journals Poliovirus 2APro Increases Viral mRNA and Polysome Stability Coordinately in Time with Cleavage of eIF4G

2008 ◽  
Vol 82 (12) ◽  
pp. 5847-5859 ◽  
Author(s):  
Brian J. Kempf ◽  
David J. Barton
Keyword(s):  
De Novo ◽  
Mrna Translation ◽  
Viral Mrna ◽  
Site Mutation ◽  
Initiation Factors ◽  
Eukaryotic Initiation Factors ◽  
Messenger Ribonucleoprotein ◽  
Infected Cells ◽  
The Stability ◽  
Polysome Stability

ABSTRACT Poliovirus (PV) 2A protease (2APro) cleaves eukaryotic initiation factors 4GI and 4GII (eIF4GI and eIF4GII) within virus-infected cells, effectively halting cap-dependent mRNA translation. PV mRNA, which does not possess a 5′ cap, is translated via cap-independent mechanisms within viral protease-modified messenger ribonucleoprotein (mRNP) complexes. In this study, we determined that 2APro activity was required for viral polysome formation and stability. 2APro cleaved eIF4GI and eIF4GII as PV polysomes assembled. A 2ACys109Ser (2APro with a Cys109Ser mutation) protease active site mutation that prevented cleavage of eIF4G coordinately inhibited the de novo formation of viral polysomes, the stability of viral polysomes, and the stability of PV mRNA within polysomes. 2ACys109Ser-associated defects in PV mRNA and polysome stability correlated with defects in PV mRNA translation. 3CPro activity was not required for viral polysome formation or stability. 2APro-mediated cleavage of eIF4G along with poly(rC) binding protein binding to the 5′ terminus of uncapped PV mRNA appear to be concerted mechanisms that allow PV mRNA to form mRNP complexes that evade cellular mRNA degradation machinery.

scholarly journals Role of Residues 121 to 124 of Vesicular Stomatitis Virus Matrix Protein in Virus Assembly and Virus-Host Interaction

2006 ◽  
Vol 80 (8) ◽  
pp. 3701-3711 ◽  
Author(s):  
John H. Connor ◽  
Margie O. McKenzie ◽  
Douglas S. Lyles
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Protein ◽  
Virus Assembly ◽  
Mrna Translation ◽  
Mutant Virus ◽  
M Protein ◽  
Viral Mrna ◽  
Wild Type ◽  
Vesicular Stomatitis ◽  
Self Association

ABSTRACT The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Analysis of the membrane association of M protein in the context of infection with isogenic recombinant viruses showed that both wild-type and mutant M proteins associated with membranes to the same extent. Virus expressing the mutant M protein did show an approximately threefold-lower binding affinity of M protein for nucleocapsid-M complexes. In contrast to the relatively minor effects of the M protein mutation on virus assembly, the mutant virus exhibited growth restriction in MDBK but not BHK cells, a slower induction of apoptosis, and lower viral-protein synthesis. Despite translating less viral protein, the mutant virus produced more viral mRNA, showing that the mutant virus could not effectively promote viral translation. These results demonstrate that the 121-to-124 region of the VSV M protein plays a minor role in virus assembly but is involved in virus-host interactions and VSV replication by augmenting viral-mRNA translation.

Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs

1986 ◽  
Vol 6 (5) ◽  
pp. 1741-1750
Author(s):  
M G Katze ◽  
B M Detjen ◽  
B Safer ◽  
R M Krug
Keyword(s):  
Protein Synthesis ◽  
Influenza Virus ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Viral Mrna ◽  
Viral Mrnas ◽  
Double Stranded Rna ◽  
Selective Translation ◽  
Infected Cells

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).

scholarly journals Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs.

1986 ◽  
Vol 6 (5) ◽  
pp. 1741-1750 ◽  
Author(s):  
M G Katze ◽  
B M Detjen ◽  
B Safer ◽  
R M Krug
Keyword(s):  
Protein Synthesis ◽  
Influenza Virus ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Viral Mrna ◽  
Viral Mrnas ◽  
Double Stranded Rna ◽  
Selective Translation ◽  
Infected Cells

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).

scholarly journals A Conserved Domain in the Leader Proteinase of Foot-and-Mouth Disease Virus Is Required for Proper Subcellular Localization and Function

2008 ◽  
Vol 83 (4) ◽  
pp. 1800-1810 ◽  
Author(s):  
Teresa de los Santos ◽  
Fayna Diaz-San Segundo ◽  
James Zhu ◽  
Marla Koster ◽  
Camila C. A. Dias ◽  
...  
Keyword(s):  
Disease Virus ◽  
Double Mutant ◽  
Foot And Mouth Disease ◽  
Mrna Translation ◽  
Mouth Disease ◽  
Mutant Virus ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Infected Cells ◽  
In Cells

ABSTRACT The leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) is involved in antagonizing the innate immune response by blocking the expression of interferon (IFN) and by reducing the immediate-early induction of IFN-β mRNA and IFN-stimulated genes. In addition to its role in shutting off cap-dependent host mRNA translation, Lpro is associated with the degradation of the p65/RelA subunit of nuclear factor κB (NF-κB). Bioinformatics analysis suggests that Lpro contains a SAP (for SAF-A/B, Acinus, and PIAS) domain, a protein structure associated in some cases with the nuclear retention of molecules involved in transcriptional control. We have introduced a single or a double mutation in conserved amino acid residues contained within this domain of Lpro. Although three stable mutant viruses were obtained, only the double mutant displayed an attenuated phenotype in cell culture. Indirect immunofluorescence analysis showed that Lpro subcellular distribution is altered in cells infected with the double mutant virus. Interestingly, nuclear p65/RelA staining disappeared from wild-type (WT) FMDV-infected cells but not from double mutant virus-infected cells. Consistent with these results, NF-κB-dependent transcription was not inhibited in cells infected with double mutant virus in contrast to cells infected with WT virus. However, degradation of the translation initiation factor eIF-4G was very similar for both the WT and the double mutant viruses. Since Lpro catalytic activity was demonstrated to be a requirement for p65/RelA degradation, our results indicate that mutation of the SAP domain reveals a novel separation-of-function activity for FMDV Lpro.

scholarly journals Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

1999 ◽  
Vol 73 (9) ◽  
pp. 7474-7488 ◽  
Author(s):  
Felicia D. Goodrum ◽  
David A. Ornelles
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Viral Mrna ◽  
Wild Type ◽  
Mrna Transport ◽  
Virus Growth ◽  
The Difference ◽  
In Cells

ABSTRACT Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G1 and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548–561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G1 cells infected with the mutant viruses compared to that in G1 cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G1. However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G1 than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G1. Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

1984 ◽  
Vol 4 (5) ◽  
pp. 966-972
Author(s):  
C Montell ◽  
E F Fisher ◽  
M H Caruthers ◽  
A J Berk
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Early Phase ◽  
Late Phase ◽  
Productive Infection ◽  
Splice Sites ◽  
Mrna Synthesis ◽  
Adenovirus E1b ◽  
Cryptic Splice Sites ◽  
Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins

1989 ◽  
Vol 9 (1) ◽  
pp. 259-267
Author(s):  
C H Agris ◽  
M E Nemeroff ◽  
R M Krug
Keyword(s):  
Specific Protein ◽  
Viral Mrna ◽  
Sequence Element ◽  
Time Points ◽  
Total Absence ◽  
Nuclear Extracts ◽  
Infected Cells ◽  
Small Nuclear Ribonucleoproteins ◽  
Rna Precursor

The assembly of mammalian pre-mRNAs into large 50S to 60S complexes, or spliceosomes, containing small nuclear ribonucleoproteins (snRNPs) leads to the production of splicing intermediates, 5' exon and lariat-3' exon, and the subsequent production of spliced products. Influenza virus NS1 mRNA, which encodes a virus-specific protein, is spliced in infected cells to form another viral mRNA (the NS2 mRNA), such that the ratio of unspliced to spliced mRNA is 10 to 1. NS1 mRNA was not detectably spliced in vitro with nuclear extracts from uninfected HeLa cells. Surprisingly, despite the almost total absence of splicing intermediates in the in vitro reaction, NS1 mRNA very efficiently formed ATP-dependent 55S complexes. The formation of 55S complexes with NS1 mRNA was compared with that obtained with an adenovirus pre-mRNA (pKT1 transcript) by using partially purified splicing fractions that restricted the splicing of the pKT1 transcript to the production of splicing intermediates. At RNA precursor levels that were considerably below saturation, approximately 10-fold more of the input NS1 mRNA than of the input pKT1 transcript formed 55S complexes at all time points examined. The pKT1 55S complexes contained splicing intermediates, whereas the NS1 55S complexes contained only precursor NS1 mRNA. Biotin-avidin affinity chromatography showed that the 55S complexes formed with either NS1 mRNA or the pKT1 transcript contained the U1, U2, U4, U5, and U6 snRNPs. Consequently, the formation of 55S complexes containing these five snRNPs was not sufficient for the catalysis of the first step of splicing, indicating that some additional step(s) needs to occur subsequent to this binding. These results indicate that the 5' splice site, 3' and branch point of NS1 and mRNA were capable of interacting with the five snRNPs to form 55S complexes, but apparently some other sequence element(s) in NS1 mRNA blocked the resolution of the 55S complexes that leads to the catalysis of splicing. On the basis of our results, we suggest mechanisms by which the splicing of NS1 is controlled in infected cells.

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