Regulation of translation initiation by herpesviruses

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.

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Rotavirus Nonstructural Protein NSP3 Is Not Required for Viral Protein Synthesis

Journal of Virology ◽

10.1128/jvi.00437-06 ◽

2006 ◽

Vol 80 (18) ◽

pp. 9031-9038 ◽

Cited By ~ 59

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4493-4498.1986 ◽

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.

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Protein Kinase R Is Responsible for the Phosphorylation of eIF2α in Rotavirus Infection

Journal of Virology ◽

10.1128/jvi.00625-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10457-10466 ◽

Cited By ~ 50

Author(s):

Margarito Rojas ◽

Carlos F. Arias ◽

Susana López

Keyword(s):

Protein Synthesis ◽

Kinase Domain ◽

Cell Protein ◽

Protein Kinase R ◽

Viral Protein Synthesis ◽

Α Subunit ◽

Rotavirus Infection ◽

Eif2α Phosphorylation ◽

Infected Cells ◽

Interferon System

ABSTRACT The eukaryotic initiation translation factor 2 (eIF2) represents a key point in the regulation of protein synthesis. This factor delivers the initiator Met-tRNA to the ribosome, a process that is conserved in all eukaryotic cells. Many types of stress reduce global translation by triggering the phosphorylation of the α subunit of eIF2, which reduces the formation of the preinitiation translation complexes. Early during rotavirus infection, eIF2α becomes phosphorylated, and even under these conditions viral protein synthesis is not affected, while most of the cell protein synthesis is blocked. Here, we found that the kinase responsible for the phosphorylation of eIF2α in rotavirus-infected cells is PKR, since in mouse embryonic fibroblasts deficient in the kinase domain of PKR, or in MA104 cells where the expression of PKR was knocked down by RNA interference, eIF2α was not phosphorylated upon rotavirus infection. The viral component responsible for the activation of PKR seems to be viral double-stranded RNA, which is found in the cytoplasm of infected cells, outside viroplasms. Taken together, these results suggest that rotaviruses induce the PKR branch of the interferon system and have evolved a mechanism to translate its proteins, surpassing the block imposed by eIF2α phosphorylation.

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

Journal of Virology ◽

10.1128/jvi.02023-07 ◽

2007 ◽

Vol 82 (2) ◽

pp. 828-839 ◽

Cited By ~ 36

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.

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The Regulation of Hepatic Protein Synthesis during Fasting in the Rat

Journal of Biological Chemistry ◽

10.1074/jbc.m410576200 ◽

2005 ◽

Vol 280 (16) ◽

pp. 16427-16436 ◽

Cited By ~ 14

Author(s):

Padmanabhan Anand ◽

Philip A. Gruppuso

Keyword(s):

Protein Synthesis ◽

Translational Control ◽

Translational Regulation ◽

Initiation Factor ◽

Pcr Analysis ◽

Mrna Levels ◽

Eif2α Phosphorylation ◽

Cap Binding Complex ◽

Branched Chain ◽

Rps6 Phosphorylation

We have studied translational control in the model of 48 h of fasting in the rat. Our initial observations showed a paradoxical increase in ribosomal protein S6 (rpS6) phosphorylation and a decrease in eukaryotic initiation factor 2α (eIF2α) phosphorylation. These effects, which would favor an increase in protein synthesis, could be attributed to increased circulating concentrations of branched-chain amino acids in fasting. To determine what mechanisms might account for decreased hepatic translation in fasting, we examined the cap binding complex. eIF4E-bound 4E-BP1 did not increase. However, eIF4E-bound eIF4G and total cellular eIF4G were profoundly decreased in fasted liver. eIF4G mRNA levels were not lower after fasting. Based on the hypothesis that decreased eIF4G translation might account for the reduced eIF4G content, we fractionated ribosomes by sucrose density centrifugation. Immunoblotting for rpS6 showed modest polysomal disaggregation upon fasting. PCR analysis of polysome profiles revealed that a spectrum of mRNAs undergo different translational regulation in the fasted state. In particular, eIF4G was minimally affected by fasting. This indicated that reduced eIF4G abundance in fasting may be a function of its stability, whereas its recovery upon refeeding is necessarily independent of its own involvement in the cap binding complex. Western immunoblotting of polysome fractions showed that phosphorylated rpS6 was disproportionately present in translating polysomes in fed and fasted animals, consistent with a role in translational control. However, the translation of rpS8, an mRNA with a 5′-oligopyrimidine tract, did not coincide with rpS6 phosphorylation, thus dissociating rpS6 phosphorylation from the translational control of this subset of mRNAs.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4493 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4493-4498 ◽

Cited By ~ 19

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.

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The role of the initiation surveillance complex in promoting efficient protein synthesis

Biochemical Society Transactions ◽

10.1042/bst0320585 ◽

2004 ◽

Vol 32 (4) ◽

pp. 585-588 ◽

Cited By ~ 10

Author(s):

D.R. Gallie

Keyword(s):

Protein Synthesis ◽

Ribosomal Subunit ◽

Initiation Factor ◽

Viral Mrnas ◽

Rate Limiting Step ◽

Complex Functions ◽

Cellular Mrnas ◽

Cap Binding Complex ◽

Regulate Protein

Initiation is most often the rate-limiting step of translation. Translation initiation requires the involvement of numerous factors that assist binding of the 40 S ribosomal subunit to an mRNA and the assembly of the 80 S ribosome at the correct initiation codon. Recruitment of an initiation surveillance complex is required for translation and serves to identify mRNAs that are structurally and functionally competent for translation. For most cellular mRNAs, recruitment of the surveillance complex requires the 5′-cap and 3′-poly(A) tail. However, some cellular and viral mRNAs that naturally lack either of these have evolved alternatives that serve to recruit the complex. The initiation surveillance complex functions to stabilize eIF4F (where eIF stands for eukaryotic initiation factor), the cap-binding complex, to the cap; promote eIF4A helicase activity to remove secondary structure in the 5′-leader that might otherwise reduce 40 S ribosomal subunit scanning; promote eIF4B binding to increase eIF4A/eIF4F function and stabilize binding of the poly(A)-binding protein to the poly(A) tail. The surveillance complex is regulated through changes in phosphorylation in response to environmental conditions or by developmental signals as a means to regulate globally protein synthesis. Thus the initiation surveillance complex ensures that only intact mRNAs are recruited for translation and serves to regulate protein synthesis.

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Regulation of mRNA Translation in Axons

The Oxford Handbook of Neuronal Protein Synthesis ◽

10.1093/oxfordhb/9780190686307.013.13 ◽

2020 ◽

Author(s):

Priyanka Patel ◽

Pabitra K. Sahoo ◽

Amar N. Kar ◽

Jeffery L. Twiss

Keyword(s):

Protein Synthesis ◽

Translational Regulation ◽

Neuronal Cell ◽

Mrna Translation ◽

Neuronal Cell Body ◽

Translational Machinery ◽

Protein Synthesis Machinery ◽

Neuronal Systems ◽

Axonal Protein ◽

Axonal Protein Synthesis

Axons can extend long distances from the neuronal cell body, and mRNA translation in axons is used to locally generate new proteins in these distal reaches of the neuron’s cytoplasm. Work over the past two decades has shown that axonal mRNA translation occurs in many different organisms and different neuronal systems. The field has progressed substantially over this time, moving from documenting mRNA translation in axons to understanding how axonal mRNA translation is regulated and what the protein products do for the neuron. Translational regulation in axons extends beyond merely controlling activity of the protein synthesis machinery. Transport of mRNAs into axons, stability of the mRNAs within the axons, and sequestration of mRNAs away from the translational machinery each contribute to determining what proteins are generated in axons, as well as when and where those proteins are generated within the axon. It is now known that thousands of different mRNAs can localize into axons. Based on unique responses to different axonal translation regulating stimuli and events, there clearly is specificity for when different mRNA populations are translated. How that specificity is driven is just now beginning to be understood, and studies emerging over the last five years point to multiple mechanisms for imparting specificity for regulation of axonal protein synthesis responses.

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Translational control of coronaviruses

Nucleic Acids Research ◽

10.1093/nar/gkaa1116 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12502-12522

Author(s):

Sylvain de Breyne ◽

Caroline Vindry ◽

Olivia Guillin ◽

Lionel Condé ◽

Fabrice Mure ◽

...

Keyword(s):

Translational Control ◽

Large Family ◽

Host Cells ◽

Translational Machinery ◽

Viral Propagation ◽

Cellular Defense ◽

Protein Synthesis Machinery ◽

Translational Apparatus

Abstract Coronaviruses represent a large family of enveloped RNA viruses that infect a large spectrum of animals. In humans, the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic and is genetically related to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), which caused outbreaks in 2002 and 2012, respectively. All viruses described to date entirely rely on the protein synthesis machinery of the host cells to produce proteins required for their replication and spread. As such, virus often need to control the cellular translational apparatus to avoid the first line of the cellular defense intended to limit the viral propagation. Thus, coronaviruses have developed remarkable strategies to hijack the host translational machinery in order to favor viral protein production. In this review, we will describe some of these strategies and will highlight the role of viral proteins and RNAs in this process.

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