Interferon-Stimulated Genes that Target Retrovirus Translation.

The innate immune system, particularly the interferon (IFN) system, constitutes the initial line of defense against viral infections. IFN signaling induces the expression of interferon-stimulated genes (ISGs), and their products frequently restrict viral infection. Retroviruses like the human immunodeficiency viruses and the human T-lymphotropic viruses cause severe human diseases and are targeted by ISG-encoded proteins. Here, we discuss ISGs that inhibit the translation of retroviral mRNAs and thereby retrovirus propagation. The Schlafen proteins degrade cellular tRNAs and rRNAs needed for translation. Zinc Finger Antiviral Protein and RNA-activated protein kinase inhibit translation initiation factors, and Shiftless suppresses translation recoding essential for the expression of retroviral enzymes. We outline common mechanisms that underlie the antiviral activity of multifunctional ISGs and discuss potential antiretroviral therapeutic approaches based on the mode of action of these ISGs.

The Inhibition of Gag-Pol Expression by the Restriction Factor Shiftless Is Dispensable for the Restriction of HIV-1 Infection.

The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.

The initiation factor 3 (IF3) residues interacting with initiator tRNA elbow modulate the fidelity of translation initiation and growth fitness in Escherichia coli.

Initiation factor 3 (IF3) regulates the fidelity of bacterial translation initiation by debarring the use of non-canonical start codons or non-initiator tRNAs and prevents premature docking of the 50S ribosomal subunit to the 30S pre-initiation complex (PIC). The C-terminal domain (CTD) of IF3 can carry out most of the known functions of IF3 and sustain Escherichia coli growth. However, the roles of the N-terminal domain (NTD) have remained unclear. We hypothesized that the interaction between NTD and initiator tRNAfMet (i-tRNA) is essential to coordinate the movement of the two domains during the initiation pathway to ensure fidelity of the process. Here, using atomistic molecular dynamics (MD) simulation, we show that R25A/Q33A/R66A mutations do not impact NTD structure but disrupt its interaction with i-tRNA. These NTD residues modulate the fidelity of translation initiation and are crucial for bacterial growth. Our observations also implicate the role of these interactions in the subunit dissociation activity of CTD of IF3. Overall, the study shows that the interactions between NTD of IF3 and i-tRNA are crucial for coupling the movements of NTD and CTD of IF3 during the initiation pathway and in imparting growth fitness to E. coli.

Mutagenic Analysis of the HIV Restriction Factor Shiftless.

The interferon-induced host cell protein shiftless (SFL) was reported to inhibit human immunodeficiency virus (HIV) infection by blocking the -1 programmed ribosomal frameshifting (-1PRF) required for expression of the Gag-Pol polyprotein. However, it is not clear how SFL inhibits -1PRF. To address this question, we focused on a 36 amino acids comprising region (termed required for antiviral activity (RAA)) that is essential for suppression of -1PRF and HIV infection and is missing from SFL short (SFLS), a splice variant of SFL with unknown function. Here, we confirm that SFL, but not SFLS, inhibits HIV -1PRF and show that inhibition is cell-type-independent. Mutagenic and biochemical analyses demonstrated that the RAA region is required for SFL self-interactions and confirmed that it is necessary for ribosome association and binding to the HIV RNA. Analysis of SFL mutants with six consecutive amino-acids-comprising deletions in the RAA region suggests effects on binding to the HIV RNA, complete inhibition of -1PRF, inhibition of Gag-Pol expression, and antiviral activity. In contrast, these amino acids did not affect SFL expression and were partially dispensable for SFL self-interactions and binding to the ribosome. Collectively, our results support the notion that SFL binds to the ribosome and the HIV RNA in order to block -1PRF and HIV infection, and suggest that the multimerization of SFL may be functionally important.

Rescuing stalled mammalian mitoribosomes - what can we learn from bacteria?

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use. The classic method for rescuing bacterial ribosomes is trans-translation. The key components of this system are absent from mammalian mitochondria; however, four members of a translation termination factor family are present, with some evidence of homology to members of a bacterial back-up rescue system. To date, there is no definitive demonstration of any other member of this family functioning in mitoribosome rescue. Here, we provide an overview of the processes and key players of canonical translation termination in both bacteria and mammalian mitochondria, followed by a perspective of the bacterial systems used to rescue stalled ribosomes. We highlight any similarities or differences with the mitochondrial translation release factors, and suggest potential roles for these proteins in ribosome rescue in mammalian mitochondria.

Translation initiation in mammalian mitochondria- a prokaryotic perspective.

ATP is generated in mitochondria of eukaryotic cells by oxidative phosphorylation (OXPHOS). The OXPHOS complex, which is crucial for cellular metabolism, comprises of both nuclear and mitochondrially encoded subunits. Also, the occurrence of several pathologies because of mutations in the mitochondrial translation apparatus indicates the importance of mitochondrial translation and its regulation. The mitochondrial translation apparatus is similar to its prokaryotic counterpart due to a common origin of evolution. However, mitochondrial translation has diverged from prokaryotic translation in many ways by reductive evolution. In this review, we focus on mammalian mitochondrial translation initiation, a highly regulated step of translation, and present a comparison with prokaryotic translation.

Sustenance of Escherichia coli on a single tRNAMet.

Living organisms possess two types of tRNAs for methionine. Initiator tRNAs bind directly into the ribosomal P-site to initiate protein synthesis, and the elongators bind to the A-site during the elongation step. Eubacterial initiators (tRNAfMet) are unique in that the methionine attached to them is formylated to facilitate their binding to initiation factor 2 (IF2), and to preclude them from binding to elongation factor Tu (EFTu). However, in mammalian mitochondria, protein synthesis proceeds with a single dual function tRNAMet. Escherichia coli possesses four tRNAfMet (initiator) and two tRNAMet (elongator) genes. Free-living organisms possessing the mitochondrion like system of single tRNAMet are unknown. We characterized mutants of E. coli tRNAfMet that function both as initiators and elongators. We show that some of the tRNAfMet mutants sustain E. coli lacking all four tRNAfMet and both tRNAMet genes, providing a basis for natural occurrence of mitochondria like situation in free living organisms. The tRNA mutants show in vivo binding to both IF2 and EFTu, indicating how they carry out these otherwise mutually exclusive functions by precise regulation of their in vivo formylation. Our results provide insights into how distinct initiator and elongator methionine tRNAs might have evolved from a single 'dual function' tRNA.

Antimicrobial activity of fusidic acid in Escherichia coli is dependent on the relative levels of ribosome recycling factor and elongation factor G.

During protein synthesis, elongation factor G (EFG) participates at the steps of translocation and ribosome recycling. Fusidic acid (FA) is a bacteriostatic antibiotic, which traps EFG on ribosomes, stalling them on mRNAs. How the bacterial susceptibility to FA is determined, and which of the two functions of EFG (translocation or ribosome recycling) is more vulnerable, has remained debatable. The in vivo studies addressing these aspects of FA mediated inhibition of protein synthesis are lacking. Here, we used a system of Escherichia coli strains and their complementation/supplementation with the plasmid borne copies of the inducible versions of EFG and ribosome recycling factor (RRF) genes. Additionally, we investigated FA sensitivity in a strain with increased proportion of stalled ribosomes. We show that the cells with high EFG/RRF (or low RRF/EFG) ratios are more susceptible to FA than those with low EFG/RRF (or high RRF/EFG) ratios. Our in vivo observations are consistent with the recent in vitro reports of dependence of FA susceptibility on EFG/RRF ratios, and the notion that an overriding target of FA is the translocation function of EFG. An applied outcome of our in vivo study is that FA mediated growth inhibition could be facilitated by depletion or inactivation of cellular RRF.

Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas.

Initiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the Escherichia coli model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host.

Fidelity of translation in the presence of mammalian mitochondrial initiation factor 3.

Initiation factor 3 (IF3) is a conserved translation factor. Mutations in mitochondrial IF3 (IF3mt) have been implicated in disease pathology. Escherichia coli infCΔ55, compromised for IF3 activity, has provided an excellent heterologous system for IF3mt structure-function analysis. IF3mt allowed promiscuous initiation from AUA, AUU and ACG codons but avoided initiation with initiator tRNAs lacking the conserved 3GC pairs in their anticodon stems. Expression of IF3mt N-terminal domain, or IF3mt devoid of its typical N-, and C-terminal extensions improved fidelity of initiation in E. coli. The observations suggest that the IF3mt terminal extensions relax the fidelity of translational initiation in mitochondria.

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits.

Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3.IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.