Translation of SARS-CoV-2 gRNA Is Extremely Efficient and Competitive despite a High Degree of Secondary Structures and the Presence of an uORF.

The SARS-CoV-2 infection generates up to nine different sub-genomic mRNAs (sgRNAs), in addition to the genomic RNA (gRNA). The 5'UTR of each viral mRNA shares the first 75 nucleotides (nt.) at their 5'end, called the leader, but differentiates by a variable sequence (0 to 190 nt. long) that follows the leader. As a result, each viral mRNA has its own specific 5'UTR in term of length, RNA structure, uORF and Kozak context; each one of these characteristics could affect mRNA expression. In this study, we have measured and compared translational efficiency of each of the ten viral transcripts. Our data show that most of them are very efficiently translated in all translational systems tested. Surprisingly, the gRNA 5'UTR, which is the longest and the most structured, was also the most efficient to initiate translation. This property is conserved in the 5'UTR of SARS-CoV-1 but not in MERS-CoV strain, mainly due to the regulation imposed by the uORF. Interestingly, the translation initiation mechanism on the SARS-CoV-2 gRNA 5'UTR requires the cap structure and the components of the eIF4F complex but showed no dependence in the presence of the poly(A) tail in vitro. Our data strongly suggest that translation initiation on SARS-CoV-2 mRNAs occurs via an unusual cap-dependent mechanism.

Translational control of coronaviruses.

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.

Focus on Translation Initiation of the HIV-1 mRNAs.

To replicate and disseminate, viruses need to manipulate and modify the cellular machinery for their own benefit. We are interested in translation, which is one of the key steps of gene expression and viruses that have developed several strategies to hijack the ribosomal complex. The type 1 human immunodeficiency virus is a good paradigm to understand the great diversity of translational control. Indeed, scanning, leaky scanning, internal ribosome entry sites, and adenosine methylation are used by ribosomes to translate spliced and unspliced HIV-1 mRNAs, and some require specific cellular factors, such as the DDX3 helicase, that mediate mRNA export and translation. In addition, some viral and cellular proteins, including the HIV-1 Tat protein, also regulate protein synthesis through targeting the protein kinase PKR, which once activated, is able to phosphorylate the eukaryotic translation initiation factor eIF2α, which results in the inhibition of cellular mRNAs translation. Finally, the infection alters the integrity of several cellular proteins, including initiation factors, that directly or indirectly regulates translation events. In this review, we will provide a global overview of the current situation of how the HIV-1 mRNAs interact with the host cellular environment to produce viral proteins.

Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame.

In the late phase of the HIV virus cycle, the unspliced genomic RNA is exported to the cytoplasm for the necessary translation of the Gag and Gag-pol polyproteins. Three distinct translation initiation mechanisms ensuring Gag production have been described with little rationale for their multiplicity. The Gag-IRES has the singularity to be located within Gag ORF and to directly interact with ribosomal 40S. Aiming at elucidating the specificity and the relevance of this interaction, we probed HIV-1 Gag-IRES structure and developed an innovative integrative modelling strategy to take into account all the gathered information. We propose a novel Gag-IRES secondary structure strongly supported by all experimental data. We further demonstrate the presence of two regions within Gag-IRES that independently and directly interact with the ribosome. Importantly, these binding sites are functionally relevant to Gag translation both in vitro and ex vivo. This work provides insight into the Gag-IRES molecular mechanism and gives compelling evidence for its physiological importance. It allows us to propose original hypotheses about the IRES physiological role and conservation among primate lentiviruses.

HIV-1 sequences isolated from patients promote expression of shorter isoforms of the Gag polyprotein.

Human immunodeficiency virus type 1 (HIV-1) unspliced mRNA drives the expression of both Gag and Gag-Pol polyproteins by using both cap- and internal ribosome entry site (IRES)-dependent translation initiation mechanisms. An IRES has been described in the matrix coding region that is involved in the production of shorter isoforms of Gag. However, up to now, this has only been shown with sequences derived from the HIV-1 laboratory strains (NL4.3 and HXB2) and never from clinical HIV-1 isolates. We have isolated ~70 sequences from HIV-1-positive patients that we have sequenced and cloned into an expression vector to monitor their ability to drive translation of Gag p55 and the shorter isoforms both in vitro and ex vivo. The results indicate that (1) the translational efficiency from the AUG-p55 varies significantly among the different isolates; (2) expression initiated at AUG-p40 codon is independent of translation initiation at the AUG-p55 triplet; and (3) all sequences promote expression of shorter Gag isoforms, in particular in Jurkat T cells, in which internal initiation occurs exclusively and directly at the AUG-p40 codon. The composition of the first ~800 nucleotides of the HIV-1 unspliced mRNA modulates the expression initiated both at the AUG-p55 and AUG-p40 codons and may impact viral production and replication. Interestingly, the AUG-p40 codon and its surrounding nucleotide context are conserved amongst clinical isolates and are used as a translation initiation site to produce a shorter Gag isoform.

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases.

UNLABELLED: Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order. IMPORTANCE: Viruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P+L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses.

Translation initiation is driven by different mechanisms on the HIV-1 and HIV-2 genomic RNAs.

The human immunodeficiency virus (HIV) unspliced full length genomic RNA possesses features of an eukaryotic cellular mRNA as it is capped at its 5' end and polyadenylated at its 3' extremity. This genomic RNA is used both for the production of the viral structural and enzymatic proteins (Gag and Pol, respectively) and as genome for encapsidation in the newly formed viral particle. Although both of these processes are critical for viral replication, they should be controlled in a timely manner for a coherent progression into the viral cycle. Some of this regulation is exerted at the level of translational control and takes place on the viral 5' untranslated region and the beginning of the gag coding region. In this review, we have focused on the different initiation mechanisms (cap- and internal ribosome entry site (IRES)-dependent) that are used by the HIV-1 and HIV-2 genomic RNAs and the cellular and viral factors that can modulate their expression. Interestingly, although HIV-1 and HIV-2 share many similarities in the overall clinical syndrome they produce, in some aspects of their replication cycle, and in the structure of their respective genome, they exhibit some differences in the way that ribosomes are recruited on the gag mRNA to initiate translation and produce the viral proteins; this will be discussed in the light of the literature.

DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs.

Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m(7)GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5' of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5' free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation.

In vitro studies reveal that different modes of initiation on HIV-1 mRNA have different levels of requirement for eukaryotic initiation factor 4F.

Expression of the two isoforms p55 and p40 of HIV-1 Gag proteins relies on distinct translation initiation mechanisms, a cap-dependent initiation and two internal ribosome entry sites (IRESs). The regulation of these processes is complex and remains poorly understood. This study was focused on the influence of the 5'-UTR and on the requirement for the eukaryotic initiation factor (eIF)4F complex components. By using an in vitro system, we showed substantial involvement of the 5'-UTR in the control of p55 expression. This highly structured 5'-UTR requires the eIF4F complex, especially RNA helicase eIF4A, which mediates initiation at the authentic AUG codon. In addition, the 5'-UTR regulates expression in an RNA concentration-dependent manner, with a high concentration of RNA triggering specific reduction of full-length Gag p55 production. HIV-1 genomic RNA also has the ability to use a strong IRES element located in the gag coding region. We show that this mechanism is particularly efficient, and that activity of this IRES is only poorly dependent on RNA helicase eIF4A when the viral 5'-UTR is removed. HIV-1 genomic mRNA exhibits in vitro translational features that allow the expression of Gag p55 protein by different mechanisms that involve different requirements for eIF4E, eIF4G, and eIF4A. This suggests that HIV-1 could adapt to its mode of translation according to the availability of the initiation factors in the infected cell.

Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites.

Viral internal ribosomal entry sites (IRESs) mediate end-independent translation initiation. There are 4 major structurally-distinct IRES groups: type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis virus), which are dissimilar except for a Yn-Xm-AUG motif at their 3' borders, type 3 (e.g., hepatitis C virus), and type 4 (dicistroviruses). Type 2-4 IRESs mediate initiation by distinct mechanisms that are nevertheless all based on specific noncanonical interactions with canonical components of the translation apparatus, such as eukaryotic initiation factor (eIF) 4G (type 2), 40S ribosomal subunits (types 3 and 4), and eIF3 (type 3). The mechanism of initiation on type 1 IRESs is unknown. We now report that domain V of type 1 IRESs, which is adjacent to the Yn-Xm-AUG motif, specifically interacts with the central domain of eIF4G. The position and orientation of eIF4G relative to the Yn-Xm-AUG motif is analogous in type 1 and 2 IRESs. eIF4G promotes recruitment of eIF4A to type 1 IRESs, and together, eIF4G and eIF4A induce conformational changes at their 3' borders. The ability of mutant type 1 IRESs to bind eIF4G/eIF4A correlated with their translational activity. These characteristics parallel the mechanism of initiation on type 2 IRESs, in which the key event is binding of eIF4G to the J-K domain adjacent to the Yn-Xm-AUG motif, which is enhanced by eIF4A. These data suggest that fundamental aspects of the mechanisms of initiation on these unrelated classes of IRESs are similar.

Cleavage of eukaryotic initiation factor eIF5B by enterovirus 3C proteases.

The enteroviruses poliovirus (PV), Coxsackie B virus (CVB) and rhinovirus (HRV) are members of Picornaviridae that inhibit host cell translation early in infection. Enterovirus translation soon predominates in infected cells, but eventually also shuts off. This complex pattern of modulation of translation suggests regulation by a multifactorial mechanism. We report here that eIF5B is proteolytically cleaved during PV and CVB infection of cultured cells, beginning at 3 hours post-infection and increasing thereafter. Recombinant PV, CVB and HRV 3Cpro cleaved purified native rabbit eukaryotic initiation factor (eIF) 5B in vitro at a single site (VVEQG, equivalent to VMEQG479 in human eIF5B) that is consistent with the cleavage specificity of enterovirus 3C proteases. Cleavage separates the N-terminal domain of eIF5B from its essential conserved central GTPase and C-terminal domains. 3Cpro-mediated cleavage of eIF5B may thus play an accessory role in the shutoff of translation that occurs in enterovirus-infected cells.

eIF2-dependent and eIF2-independent modes of initiation on the CSFV IRES: a common role of domain II.

Specific interactions of the classical swine fever virus internal ribosomal entry site (IRES) with 40S ribosomal subunits and eukaryotic translation initiation factor (eIF)3 enable 43S preinitiation complexes containing eIF3 and eIF2-GTP-Met-tRNA(iMet) to bind directly to the initiation codon, yielding 48S initiation complexes. We report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(iMet) binding to IRES-40S complexes, forming 48S complexes that can assemble elongation-competent ribosomes. Although 48S complexes assembled both by eIF2/eIF3- and eIF5B/eIF3-mediated Met-tRNA(iMet) recruitment were destabilized by eIF1, dissociation of 48S complexes formed with eIF2 could be out-competed by efficient subunit joining. Deletion of IRES domain II, which is responsible for conformational changes induced in 40S subunits by IRES binding, eliminated the sensitivity of 48S complexes assembled by eIF2/eIF3- and eIF5B/eIF3-mediated mechanisms to eIF1-induced destabilization. However, 48S complexes formed by the eIF5B/eIF3-mediated mechanism on the truncated IRES could not undergo efficient subunit joining, as reported previously for analogous complexes assembled with eIF2, indicating that domain II is essential for general conformational changes in 48S complexes, irrespective of how they were assembled, that are required for eIF5-induced hydrolysis of eIF2-bound GTP and/or subunit joining.

Factor requirements for translation initiation on the Simian picornavirus internal ribosomal entry site.

The Simian picornavirus type 9 (SPV9) 5'-untranslated region (5' UTR) has been predicted to contain an internal ribosomal entry site (IRES) with structural elements that resemble domains of hepacivirus/pestivirus (HP) IRESs. In vitro reconstitution of initiation confirmed that this 5' UTR contains an IRES and revealed that it has both functional similarities and differences compared to HP IRESs. Like HP IRESs, the SPV9 IRES bound directly to 40S subunits and eukaryotic initiation factor (eIF) 3, depended on the conserved domain IIId for ribosomal binding and consequently for function, and additionally required eIF2/initiator tRNA to yield 48S complexes that formed elongation-competent 80S ribosomes in the presence of eIF5, eIF5B, and 60S subunits. Toeprinting analysis revealed that eIF1A stabilized 48S complexes, whereas eIF1 induced conformational changes in the 40S subunit, likely corresponding to partial opening of the entry latch of the mRNA-binding channel, that were exacerbated by eIF3 and suppressed by eIF1A. The SPV9 IRES differed from HP IRESs in that its function was enhanced by eIF4A/eIF4F when the IRES was adjacent to the wild-type coding sequence, but was less affected by these factors or by a dominant negative eIF4A mutant when potentially less structured coding sequences were present. Exceptionally, this IRES promoted binding of initiator tRNA to the initiation codon in the P site of 40S subunits independently of eIF2. Although these 40S/IRES/tRNA complexes could not form active 80S ribosomes, this constitutes a second difference between the SPV9 and HP IRESs. eIF1 destabilized the eIF2-independent ribosomal binding of initiator tRNA.

In vitro reconstitution and biochemical characterization of translation initiation by internal ribosomal entry.

The internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV) and related viruses promote initiation of translation by a noncanonical end-independent mechanism. To characterize this mechanism at the molecular level, we have developed biochemical approaches to reconstitute the process in vitro from individual purified components of the translation apparatus, developed methods to characterize steps in this process so that the functions of individual proteins can be characterized, and adapted assays such as primer extension inhibition ("toe printing") to monitor accurate assembly on the IRES of ribosomal 48S and 80S complexes. In vitro reconstitution of 48S complex formation offers an approach for the functional identification of IRES trans-acting factors (ITAFs) that are required for initiation in addition to canonical initiation factors and revealed that despite being related, different EMCV-like IRESs nevertheless have distinct ITAF requirements. Toe printing revealed that a common feature of initiation on EMCV-like IRESs is the stable binding of an eIF4G/eIF4A complex to them near the initiation codon, where it can locally unwind RNA to facilitate ribosomal attachment. The same toe printing assay indicated that binding of ITAFs to these IRESs enhances binding of these two canonical initiation factors. We also describe protocols for chemical and enzymatic footprinting to determine the interactions of trans-acting factors with the IRES at nucleotide resolution and for directed hydroxyl radical probing to determine their orientation on the IRES.

A distinct group of hepacivirus/pestivirus-like internal ribosomal entry sites in members of diverse picornavirus genera: evidence for modular exchange of functional noncoding RNA elements by recombination.

The 5' untranslated regions (UTRs) of the RNA genomes of Flaviviridae of the Hepacivirus and Pestivirus genera contain internal ribosomal entry sites (IRESs) that are unrelated to the two principal classes of IRESs of Picornaviridae. The mechanism of translation initiation on hepacivirus/pestivirus (HP) IRESs, which involves factor-independent binding to ribosomal 40S subunits, also differs fundamentally from initiation on these picornavirus IRESs. Ribosomal binding to HP IRESs requires conserved sequences that form a pseudoknot and the adjacent IIId and IIIe domains; analogous elements do not occur in the two principal groups of picornavirus IRESs. Here, comparative sequence analysis was used to identify a subset of picornaviruses from multiple genera that contain 5' UTR sequences with significant similarities to HP IRESs. They are avian encephalomyelitis virus, duck hepatitis virus 1, duck picornavirus, porcine teschovirus, porcine enterovirus 8, Seneca Valley virus, and simian picornavirus. Their 5' UTRs are predicted to form several structures, in some of which the peripheral elements differ from the corresponding HP IRES elements but in which the core pseudoknot, domain IIId, and domain IIIe elements are all closely related. These findings suggest that HP-like IRESs have been exchanged between unrelated virus families by recombination and support the hypothesis that RNA viruses consist of modular coding and noncoding elements that can exchange and evolve independently.

Intracellular processing of the Sendai virus C' protein leads to the generation of a Y protein module: structure-functional implications.

The Sendai virus "C-proteins" (C', C, Y1 and Y2) are a nested set of non-structural proteins. The shorter Y proteins arise in vivo both by de novo translation initiation and by proteolytic processing of C'. In this paper, we demonstrate that C' but not C (differing only by 11 N-terminal amino acid) serves as an efficient substrate for intracellular processing. However, processing can be mimicked in vitro by the addition of endopeptidases. Under conditions of limited proteolysis we observed that in a fraction of the C' protein the Y region exists as a proteinase resistant core. This core was conserved in the C protein. We propose that C' functions as a Pro-protein delivering the Y module to a specific intracellular location.

Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement.

The genomic RNA of the gypsy retroelement from Drosophila melanogaster exhibits features similar to other retroviral RNAs because its 5' untranslated (5' UTR) region is unusually long (846 nucleotides) and potentially highly structured. Our initial aim was to search for an internal ribosome entry site (IRES) element in the 5' UTR of the gypsy genomic RNA by using various monocistronic and bicistronic RNAs in the rabbit reticulocyte lysate (RRL) system and in cultured cells. Results reported here show that two functionally distinct and independent RNA domains control the production of gypsy encoded proteins. The first domain corresponds to the 5' UTR of the env subgenomic RNA and exhibits features of an efficient IRES (IRES(E)) both in the reticulocyte lysate and in cells. The second RNA domain that encompasses the gypsy insulator can function as an IRES in the rabbit reticulocyte lysate but strongly represses translation in cultured cells. Taken together, these results suggest that expression of the gypsy encoded proteins from the genomic and subgenomic RNAs can be regulated at the level of translation.

Proteolytic processing and translation initiation: two independent mechanisms for the expression of the Sendai virus Y proteins.

The four Sendai virus C-proteins (C', C, Y1, and Y2) represent an N-terminal nested set of non-structural proteins whose expression modulates both the readout of the viral genome and the host cell response. In particular, they modulate the innate immune response by perturbing the signaling of type 1 interferons. The initiation codons for the four C-proteins have been mapped in vitro, and it has been proposed that the Y proteins are initiated by ribosomal shunting. A number of mutations were reported that significantly enhanced Y expression, and this was attributed to increased shunt-mediated initiation. However, we demonstrate that this arises due to enhanced proteolytic processing of C', an event that requires its very N terminus. Curiously, although Y expression in vitro is mediated almost exclusively by initiation, Y proteins in vivo can arise both by translation initiation and processing of the C' protein. To our knowledge this is the first example of two apparently independent pathways leading to the expression of the same polypeptide chain. This dual pathway explains several features of Y expression.

Identification of a cis-acting element required for shunt-mediated translational initiation of the Sendai virus Y proteins.

Shunting is a mechanism that permits translational initiation at internal codons positioned in proximity to a ribosome acceptor sequence. Sendai virus exploits shunting to express a series of proteins that initiate at the fourth and fifth start sites on the P/C mRNA (namely, the Y1 and Y2 proteins, respectively). Shunt-mediated initiation at these sites is codon independent. In an attempt to characterise the acceptor site, an extensive deletion analysis was performed spanning the entire C ORF. Only mutants flanking the Y1/Y2 start sites exhibited altered shunt phenotypes. Some of these significantly enhanced shunting efficiency to the point where the Y1/Y2 proteins were the major translational products of the mRNA. Additionally, removal of a short region just downstream of the Y2 start codon (referred to as Delta10) ablated all Y protein initiation via shunting but had no effect on Y expression when the AUG codons were viewed by a scanning ribosome. Point mutations introduced into this Delta10 sequence severely perturbed shunt-mediated initiation. We also provide evidence that changes in this region of the P/C mRNA may be used to modulate Y protein expression levels in different viral strains.