An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines

1986 ◽  
Vol 6 (12) ◽  
pp. 4493-4498
Author(s):  
J Kitajewski ◽  
R J Schneider ◽  
B Safer ◽  
T Shenk
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Rna Polymerase Iii ◽  
Growth Responses ◽  
Kb Cells ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
Translational Initiation ◽  
Cell Extracts ◽  
293 Cells

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.

scholarly journals An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines.

1986 ◽  
Vol 6 (12) ◽  
pp. 4493-4498 ◽  
Author(s):  
J Kitajewski ◽  
R J Schneider ◽  
B Safer ◽  
T Shenk
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Rna Polymerase Iii ◽  
Growth Responses ◽  
Kb Cells ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
Translational Initiation ◽  
Cell Extracts ◽  
293 Cells

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.

scholarly journals KSHV lytic mRNA is efficiently translated in the absence of eIF4F

2018 ◽  
Author(s):  
Eric S. Pringle ◽  
Carolyn-Ann Robinson ◽  
Nicolas Crapoulet ◽  
Andrea L-A. Monjo ◽  
Katrina Bouzanis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Host Cell ◽  
Translation Initiation ◽  
Viral Protein ◽  
Viral Proteins ◽  
Lytic Replication ◽  
Cell Protein ◽  
Viral Protein Synthesis ◽  
Viral Mrnas

ABSTRACTHerpesvirus genomes are decoded by host RNA polymerase II, generating messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5 ′ -m7GTP caps and poly(A) tails that should permit assembly of canonical eIF4F cap-binding complexes to initiate protein synthesis. However, we have shown that chemical disruption of eIF4F does not impede KSHV lytic replication, suggesting that alternative translation initiation mechanisms support viral protein synthesis. Here, using polysome profiling analysis, we confirmed that eIF4F disassembly did not affect the efficient translation of viral mRNAs during lytic replication, whereas a large fraction of host mRNAs remained eIF4F-dependent. Lytic replication altered multiple host translation initiation factors (TIFs), causing caspase-dependent cleavage of eIF2α and eIF4G1 and decreasing levels of eIF4G2 and eIF4G3. Non-eIF4F TIFs NCBP1, eIF4E2 and eIF4G2 associated with actively translating messenger ribonucleoprotein (mRNP) complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production, suggesting that the virus does not exclusively rely on one of these alternative TIFs for efficient viral protein synthesis. METTL3, an N6-methyladenosine (m6A) methyltransferase that modifies mRNAs and influences translational efficiency, was dispensable for early viral gene expression and genome replication but required for late gene expression and virion production. METTL3 was also subject to caspase-dependent degradation during lytic replication, suggesting that its positive effect on KSHV late gene expression may be indirect. Taken together, our findings reveal extensive remodelling of TIFs during lytic replication, which may help sustain efficient viral protein synthesis in the context of host shutoff.IMPORTANCEViruses use host cell protein synthesis machinery to create viral proteins. Herpesviruses have evolved a variety of ways to gain control over this host machinery to ensure priority synthesis of viral proteins and diminished synthesis of host proteins with antiviral properties. We have shown that a herpesvirus called KSHV disrupts normal cellular control of protein synthesis. A host cell protein complex called eIF4F starts translation of most cellular mRNAs, but we observed it is dispensable for efficient synthesis of viral proteins. Several proteins involved in alternative modes of translation initiation were likewise dispensable. However, an enzyme called METTL3 that modifies mRNAs is required for efficient synthesis of certain late KSHV proteins and productive infection. We observed caspase-dependent degradation of several host cell translation initiation proteins during infection, suggesting that the virus alters pools of available factors to favour efficient viral protein synthesis at the expense of host protein synthesis.

Regulation of translation initiation by herpesviruses

2008 ◽  
Vol 36 (4) ◽  
pp. 701-707 ◽  
Author(s):  
Richard W.P. Smith ◽  
Sheila V. Graham ◽  
Nicola K. Gray
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Translational Regulation ◽  
Large Family ◽  
Cell Protein ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
Dna Viruses ◽  
Protein Synthesis Machinery ◽  
Regulate Protein

Viruses are dependent upon the host cell protein synthesis machinery, thus they have developed a range of strategies to manipulate host translation to favour viral protein synthesis. Consequently, the study of viral translation has been a powerful tool for illuminating many aspects of cellular translational control. Although much work to date has focused on translational regulation by RNA viruses, DNA viruses have also evolved complex mechanisms to regulate protein synthesis. Here we summarize work on a large family of DNA viruses, the Herpesviridae, which have evolved mechanisms to sustain efficient cap-dependent translation and to regulate the translation of specific viral mRNAs.

scholarly journals Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation

2007 ◽  
Vol 82 (2) ◽  
pp. 828-839 ◽  
Author(s):  
Maria D. Gainey ◽  
Patrick J. Dillon ◽  
Kimberly M. Clark ◽  
Mary J. Manuse ◽  
Griffith D. Parks
Keyword(s):  
Protein Synthesis ◽  
Hela Cells ◽  
Viral Protein ◽  
Time Course ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
V Protein

ABSTRACT The paramyxovirus simian virus 5 (SV5) establishes highly productive persistent infections of epithelial cells without inducing a global inhibition of translation. Here we show that an SV5 mutant (the P/V-CPI− mutant) with substitutions in the P subunit of the viral polymerase and the accessory V protein also establishes highly productive infections like wild-type (WT) SV5 but that cells infected with the P/V-CPI− mutant show an overall shutdown of both host and viral translation at late times postinfection. Reduced host and viral protein synthesis with the P/V-CPI− virus was not due to lower levels of mRNA or caspase-dependent apoptosis and correlated with phosphorylation of the translation initiation factor eIF-2α. WT SV5 was a poor activator of the eIF-2α kinase protein kinase R (PKR). By contrast, the P/V-CPI− mutant induced PKR phosphorylation, which correlated with the time course of translation inhibition but was independent of interferon signaling. In HeLa cells that expressed the PKR inhibitor influenza A virus NS1 or reovirus sigma3, the rate of host protein synthesis at late times after infection with the P/V-CPI− mutant was restored to ∼50% that of control HeLa cells. By contrast, the rates of P/V-CPI− viral protein synthesis in HeLa cells expressing NS1 or sigma3 were dramatically enhanced, between 5- and 20-fold, while levels of viral mRNA were increased only slightly (NS1-expressing cells) or remained constant (sigma3-expressing cells). Similar results were found using HeLa cells where PKR levels were reduced due to knockdown by small interfering RNA. Expression of either the WT P or the WT V protein from the genome of the P/V-CPI− mutant resulted in lower levels of PKR activation and rates of host and viral protein synthesis that closely matched those seen with WT SV5. Despite higher rates of translation, cells infected with the V- or P-complemented virus accumulated viral mRNAs to lower levels than that seen with the parental P/V-CPI− mutant. We present a model in which the paramyxovirus P/V gene products limit induction of PKR by limiting the synthesis of aberrant viral mRNAs and double-stranded RNA and thus prevent the shutdown of translation by a mechanism that differs from that of other PKR inhibitors such as NS1 and sigma3.

scholarly journals Rotavirus Nonstructural Protein NSP3 Is Not Required for Viral Protein Synthesis

2006 ◽  
Vol 80 (18) ◽  
pp. 9031-9038 ◽  
Author(s):  
Hilda Montero ◽  
Carlos F. Arias ◽  
Susana Lopez
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Nonstructural Protein ◽  
Consensus Sequence ◽  
Initiation Factor ◽  
Cell Protein ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
Initiation Of Translation ◽  
Cellular Mrnas

ABSTRACT Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5′ end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3′ end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3′ ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.

scholarly journals Human Enterovirus Group B Viruses Rely on Vimentin Dynamics for Efficient Processing of Viral Nonstructural Proteins

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Paula Turkki ◽  
Mira Laajala ◽  
Malin Flodström-Tullberg ◽  
Varpu Marjomäki
Keyword(s):  
Protein Synthesis ◽  
Cell Survival ◽  
Host Cell ◽  
Viral Protein ◽  
Viral Proteins ◽  
Structural Proteins ◽  
Human Enterovirus ◽  
Nonstructural Proteins ◽  
Viral Protein Synthesis ◽  
Group B

ABSTRACT We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with β, β′-iminodipropionitrile (IDPN) did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the nonstructural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, smaller amounts of nonstructural proteins did not result from proteasomal degradation but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1 but had less effect on 2A. The results suggest that the vimentin dynamics regulate viral nonstructural protein synthesis while having less effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and nonstructural proteins of enteroviruses, having consequences on host cell survival. IMPORTANCE A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and nonstructural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the nonstructural proteins orchestrate the takeover of the host cell and its functions. Here, we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages.

scholarly journals Origin of the Eukaryotic Cell

2017 ◽  
Vol 01 (02) ◽  
pp. 108-120 ◽  
Author(s):  
Nick Lane
Keyword(s):  
Protein Synthesis ◽  
Host Cell ◽  
Genome Size ◽  
Energy Savings ◽  
Nuclear Genome ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Bacterial Genomes ◽  
Complex Cells ◽  
Life On Earth

All complex life on Earth is composed of ‘eukaryotic’ cells. Eukaryotes arose just once in 4 billion years, via an endosymbiosis — bacteria entered a simple host cell, evolving into mitochondria, the ‘powerhouses’ of complex cells. Mitochondria lost most of their genes, retaining only those needed for respiration, giving eukaryotes ‘multi-bacterial’ power without the costs of maintaining thousands of complete bacterial genomes. These energy savings supported a substantial expansion in nuclear genome size, and far more protein synthesis from each gene.

Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat

1986 ◽  
Vol 6 (6) ◽  
pp. 2041-2052
Author(s):  
C Perez-Stable ◽  
C K Shen
Keyword(s):  
Hela Cell ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Promoter Element ◽  
Class Iii ◽  
Promoter Elements ◽  
Transcriptional Initiation ◽  
Cell Extracts ◽  
Alu Repeat ◽  
Factor C

Similar to tRNA genes and the VAI gene, the Alu family repeats are transcribed by RNA polymerase III and contain a split intragenic promoter. Results of our previous studies have shown that when the anterior, box A-containing promoter element (5'-Pu-Pu-Py-N-N-Pu-Pu-Py-G-G-3' in which Pu is any purine, Py is any pyrimidine, and N is any nucleotide) of a human Alu family repeat is deleted, the remaining box B-containing promoter element (5'-G-A/T-T-C-Pu-A-N-N-C-3') is still capable of directing weak transcriptional initiation at approximately 70 base pairs (bp) upstream from the box B sequence. This is different from the tRNA genes in which the box A-containing promoter element plays the major role in the positioning of the transcriptional initiation site(s). To account for this difference, we first carried out competition experiments in which we show that the posterior element of the Alu repeat competes with the VAI gene effectively for the transcription factor C in HeLa cell extracts. We then constructed a series of contraction and expansion mutants of the Alu repeat promoter in which the spacing between boxes A and B was systematically varied by molecular cloning. In vitro transcription of these clones in HeLa cell extracts was analyzed by RNA gel electrophoresis and primer extension mapping. We show that when the box A and box B promoter sequences are separated by 47 to 298 bp, the transcriptional initiation sites remain 4 to 5 bp upstream from box A. However, this positioning function by the box A-containing promoter element was lost when the spacing was shortened to only 26 bp or increased to longer than 600 bp. Instead, transcriptional initiation occurred approximately 70 bp upstream from box B, similar to that in the clones containing only the box B promoter element. All the mutant clones were transcribed less efficiently than was the wild type. An increase in the distance between boxes A and B also activated a second box A-like element within the Alu family repeat. We compare these results with the results of tRNA gene studies. We also discuss this comparison in terms of the positioning function of the split class III promoter elements and the evolutionary conservation of the spacing between the two promoter elements for optimum transcriptional efficiency.

Sign in / Sign up

Export Citation Format

Share Document