The lifecycle of retroviruses and retrotransposons includes a reverse transcription step, wherein dsDNA is synthesized from genomic RNA for subsequent insertion into the host genome. Retroviruses and retrotransposons commonly appropriate major components of the host cell translational machinery, including cellular tRNAs, which are exploited as reverse transcription primers. Nonpriming functions of tRNAs have also been proposed, such as in HIV-1 virion assembly, and tRNAderived fragments may also be involved in retrovirus and retrotransposon replication. Moreover, host cellular proteins regulate retroviral replication by binding to tRNAs and thereby affecting various steps in the viral lifecycle. For example, in some cases, tRNA primer selection is facilitated by cognate aminoacyl-tRNA synthetases (ARSs), which bind tRNAs and ligate them to their corresponding amino acids, but also have many known nontranslational functions. Multi-omic studies have revealed that ARSs interact with both viral proteins and RNAs and potentially regulate retroviral replication. Here, we review the currently known roles of tRNAs and their derivatives in retroviral and retrotransposon replication and shed light on the roles of tRNA-binding proteins such as ARSs in this process. Figure 2. HIV-1 tRNA primer recruitment and packaging. HIV-1 infection induces a pool of free, non-MSC-associated phosphorylated LysRS, which traffics to the nucleus. The role of nuclear LysRS is unknown. The genomic RNA/Gag/Gag-Pol complex recruits the LysRS/tRNA complex. The TLE binds LysRS and releases the tRNA primer for annealing. The cellular location of tRNA primer annealing is unknown but is depicted here in the cytoplasm. The PBS and anti-PBS region of the tRNA are colored in red. The TLE and the tRNA region mimicked are colored in green.JBC REVIEWS: tRNA and synthetases in retroviral replication
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The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly in vitro with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity. L ate in the retroviral life cycle, the structural protein Gag localizes to the inner leaflet of the plasma membrane (PM), where it assembles around the viral genomic RNA, leading to budding from the cell. Gag proteins include three major domains, the Nterminal matrix (MA) domain, which mediates membrane binding; the central capsid (CA) domain, which mediates Gag-Gag interactions during virion assembly; and the C-terminal nucleocapsid (NC...
Aminoacyl-tRNA synthetases are enzymes that ensure accurate protein synthesis.Variants of the dual-functional cytoplasmic human glutamyl-prolyl-tRNA synthetase, EPRS1, have been associated with leukodystrophy, diabetes and bone disease. Here, we report compound heterozygous variants in EPRS1 in a 4-year-old female patient presenting with psychomotor developmental delay, seizures and deafness. Functional studies of these two missense mutations support major defects in enzymatic function in vitro and contributed to confirmation of the diagnosis.
Host factor tRNAs facilitate the replication of retroviruses such as human immunodeficiency virus type 1 (HIV-1). HIV-1 uses human tRNALys3 as the primer for reverse transcription, and the assembly of HIV-1 structural protein Gag at the plasma membrane (PM) is regulated by matrix (MA) domain–tRNA interactions. A large, dynamic multi-aminoacyl-tRNA synthetase complex (MSC) exists in the cytosol and consists of eight aminoacyl-tRNA synthetases (ARSs) and three other cellular proteins. Proteomic studies to identify HIV–host interactions have identified the MSC as part of the HIV-1 Gag and MA interactomes. Here, we confirmed that the MA domain of HIV-1 Gag forms a stable complex with the MSC, mapped the primary interaction site to the linker domain of bi-functional human glutamyl-prolyl-tRNA synthetase (EPRS), and showed that the MA–EPRS interaction was RNA dependent. MA mutations that significantly reduced the EPRS interaction reduced viral infectivity and mapped to MA residues that also interact with phosphatidylinositol-(4,5)-bisphosphate. Overexpression of EPRS or EPRS fragments did not affect susceptibility to HIV-1 infection, and knockdown of EPRS reduced both a control reporter gene and HIV-1 protein translation. EPRS knockdown resulted in decreased progeny virion production, but the decrease could not be attributed to selective effects on virus gene expression, and the specific infectivity of the virions remained unchanged. While the precise function of the Gag–EPRS interaction remains uncertain, we discuss possible effects of the interaction on either virus or host activities.
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