Adam P. Geballe

Upstream open reading frames as regulators of mRNA translation.

Continuing discoveries of new and surprising mechanisms of gene regulation suggest that our understanding of this complex and ubiquitous biological process remains incomplete. Emerging examples illustrate that many and perhaps all genes are regulated at multiple steps including transcription, posttranscriptional processing, nuclear export and localization, stability, and translation of mature mRNA molecules. Translation itself is regulated by a diverse collection of mechanisms that act not only at the initiation step but also during elongation and termination and even after termination. Among the various cis elements in mRNAs (43) that participate in regulating translation are AUG codons within transcript leaders (upstream AUGs [uAUGs]) and, in some cases, associated upstream open reading frames (uORFs). Based on a 1987 survey, less than 10% of eukaryotic mRNAs contain AUG codons within their transcript leader regions (often erroneously referred to as 5′ untranslated regions). However, uAUGs are conspicuously common in certain classes of genes, including two-thirds of oncogenes and many other genes involved in the control of cellular growth and differentiation (29, 31, 42). Despite the wealth of sequence data being generated by large-scale sequencing projects, extracting an up-to-date, comprehensive, and accurate estimate of the number of genes with uORFs is a formidable task. Only a minority of database entries are based on careful mRNA mapping data with annotations that identify the precise start of the transcript leader. Moreover, the use of alternative transcriptional start sites, alternative RNA processing, and alternative initiation codons complicates the determination of what exactly constitutes the transcript leader. Nonetheless, it is clear that uAUGs are not uncommon in genes with critical cellular roles, and identifying when and how they function is necessary if we are to achieve a comprehensive understanding of the interesting genes that contain these elements and of eukaryotic gene regulation in general. Some of the general principles by which uORFs participate in translational control are beginning to be understood. In this article, we first review these principles, which include the process of recognition of uORFs, regulation of reinitiation at downstream cistrons after translation of uORFs, and regulatory effects of peptides encoded by uORFs. We then illustrate how these principles are applied by reviewing several specific examples where the roles of uORFs in translational control have been well characterized.

Cytomegalovirus: pathogen, paradigm, and puzzle.

Human cytomegalovirus (CMV), one of the eight herpesviruses that commonly infect humans, is best known for its propensity to cause disease in immunocompromised patients, especially transplant recipients, patients with advanced AIDS, and congenitally infected newborns. Advances in molecular virology coupled with improvements in diagnostic methods and treatment options have vastly improved our understanding of and ability to manage CMV, but many uncertainties remain, including the mechanisms of persistence and pathogenesis and its hypothesized roles in a variety of human illnesses. Here we review recent advances that are reshaping our view and approach to this fascinating virus.

Protein kinase R reveals an evolutionary model for defeating viral mimicry

Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis and cytoskeletal dynamics. Other mimics interfere with immunity. Poxviruses encode K3L, a mimic of eIF2α, which is the substrate of protein kinase R (PKR), an important component of innate immunity in vertebrates. The PKR–K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2α) while avoiding rapidly evolving substrate mimics such as K3L. Using the PKR–K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under intense episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2α recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, although it can seem that pathogens gain insurmountable advantages by mimicking cellular components, host factors such as PKR can compete in molecular ‘arms races’ with mimics because of evolutionary flexibility at protein interaction interfaces challenged by mimicry.

Evasion of Cellular Antiviral Responses by Human Cytomegalovirus TRS1 and IRS1

ABSTRACT During infection with human cytomegalovirus (HCMV), cellular protein synthesis continues even as viral proteins are being synthesized in abundance. Thus, HCMV may have a mechanism for counteracting host cell antiviral pathways that act by shutting off translation. Consistent with this view, HCMV infection of human fibroblasts rescues the replication of a vaccinia virus mutant lacking the double-stranded RNA-binding protein gene E3L (VVΔE3L). HCMV also prevents the phosphorylation of the eukaryotic translation initiation factor eIF-2α, the activation of RNase L, and the shutoff of viral and cellular protein synthesis that otherwise result from VVΔE3L infection. To identify the HCMV gene(s) responsible for these effects, we prepared a library of VVΔE3L recombinants containing HCMV genomic fragments. By infecting nonpermissive cells with this library and screening for VV gene expression and replication, we isolated a virus containing a 2.8-kb HCMV fragment that rescues replication of VVΔE3L. The fragment comprises the 3′ end of the J1S open reading frame through the entire TRS1 gene. Analyses of additional VVΔE3L recombinants revealed that the protein encoded by TRS1, pTRS1, as well as the closely related IRS1 gene, rescues VVΔE3L replication and prevent the shutoff of protein synthesis, the phosphorylation of eIF-2α, and activation of RNase L. These results demonstrate that TRS1 and IRS1 are able to counteract critical host cell antiviral response pathways.

Translational Control by an Upstream Open Reading Frame in the HER-2/neu Transcript

Overexpression of the HER-2 (neu,erbB-2) receptor results in cellular transformation and is associated with a variety of human cancers. Multiple mechanisms, including gene amplification and transcriptional, post-transcriptional, and translational controls contribute to the regulation of HER-2 expression. One of the components of these regulatory mechanisms is a short upstream open reading frame (uORF) in the HER-2 mRNA that represses downstream translation in a variety of cell types. Here we explore the mechanism by which this uORF exerts its inhibitory effect. As judged by comparisons of protein and mRNA abundance and by polysomal distribution analyses, the uORF represses translation of the HER-2 cistron or of a heterologous reporter gene. Despite its conservation among mammalian species, the peptide sequence of the uORF is not required for this inhibitory effect. Rather, the majority of ribosomes that load on the HER-2 mRNA most likely translate the uORF and are then unable to reinitiate at the downstream AUG codon, in part due to the short intercistronic spacing. A minority of ribosomes gain access to the HER-2 initiation codon either by leaky scanning past the upstream AUG codon or by reinitiating after having translated the uORF despite the short intercistronic region. These results suggest that the HER-2 uORF controls synthesis of this oncoprotein by limiting ribosomal access to downstream initiation sites.

The Human Cytomegalovirus Protein TRS1 Inhibits Autophagy via Its Interaction with Beclin 1

ABSTRACT Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stimulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect on macroautophagy. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P. Codogno, and A. Esclatine, Autophagy 4:1–8, 2008) by a mechanism that requires de novo viral protein expression. Exploration of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Although this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2α by PKR has been described as a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

ABSTRACT The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVΔE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2α kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVΔE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVΔE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVΔE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVΔE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2α.

CODING SEQUENCE-DEPENDENT RIBOSOMAL ARREST AT TERMINATION OF TRANSLATION

A remarkably high percentage of proto-oncogene, growth factor, cellular receptor, and viral transcript leaders contain short upstream open reading frames (uORFs), yet the significance and regulatory effects of these uORFs have not been well characterized. In the case of the human cytomegalovirus gpUL4 (gp48) transcript, the second of three uORFs (uORF2) inhibits translation of the downstream cistron by a process that depends on the uORF2 amino acid coding information. To investigate the mechanism underlying this unusual regulatory element, we adapted the toeprinting (or reverse transcriptase extension inhibition) assay for use in detecting positions of ribosomal stalling on gp48 transcripts. Using a cell-free translation system, we demonstrate that ribosomes arrest at the termination codon of uORF2 by a uORF2 coding sequence-dependent mechanism. Further, the sequence requirements for ribosomal stalling are the same as for inhibition of downstream translation. We also provide evidence for ribosomal stalling in vivo, on the natural viral mRNA. These data support the hypothesis that the inhibition of downstream translation results from uORF2 peptide-dependent ribosomal arrest at termination and suggest that translation termination may be a regulatory step in expression of some eukaryotic genes.

Essential Role for either TRS1 or IRS1 in Human Cytomegalovirus Replication

ABSTRACT Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses, including the global repression of protein synthesis mediated by protein kinase R (PKR) and 2′-5′ oligoadenylate synthetase (OAS). As a consequence, many viruses have evolved genes, such as those encoding dsRNA-binding proteins, which counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pTRS1 and pIRS1, which bind dsRNA and can prevent activation of the PKR and OAS pathways. HCMV mutants lacking either IRS1 or TRS1 replicate at least moderately well in cell culture. However, as we demonstrate in the present study, an HCMV mutant lacking both IRS1 and TRS1 (HCMV[ΔI/ΔT]) has a severe replication defect. Infection with HCMV[ΔI/ΔT] results in a profound inhibition of overall and viral protein synthesis, as well as increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). The vaccinia virus E3L gene can substitute for IRS1 or TRS1, enabling HCMV replication. Despite the accumulation of dsRNA in HCMV-infected cells, the OAS pathway remains inactive, even in HCMV[ΔI/ΔT]-infected cells. These results suggest that PKR-mediated phosphorylation of eIF2α is the dominant dsRNA-activated pathway responsible for inhibition of protein synthesis and HCMV replication in the absence of both IRS1 and TRS1 and that the requirement for evasion of the PKR pathway likely explains the necessity for IRS1 or TRS1 for productive infection.

Inhibition of Translation Termination Mediated by an Interaction of Eukaryotic Release Factor 1 with a Nascent Peptidyl-tRNA

ABSTRACT Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.