HIV-1, like all retroviruses, irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the Strand Transfer Complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. Here, using a soluble IN fusion protein, we present a high-resolution cryo-electron microscopy (cryoEM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts distinct domain rearrangements. The unique manner of STC assembly highlights how HIV-1 can use the common retroviral intasome core architecture to accommodate different IN domain modules for integration.

The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretroviral drugs, integrase strand-transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo–electron microscopy structures of HIV intasomes bound to the latest generation of INSTIs. These structures highlight how small changes in the integrase active site can have notable implications for drug binding and design and provide mechanistic insights into why a leading INSTI retains efficacy against a broad spectrum of drug-resistant variants. The data have implications for expanding effective treatments available for HIV-infected individuals.

Eukaryotic mRNAs are subject to quality control mechanisms that degrade defective mRNAs. In yeast, mRNAs with stalls in translation elongation are targeted for endonucleolytic cleavage by No-Go decay (NGD). The cleavage triggered by No-Go decay is dependent on Dom34p and Hbs1p, and Dom34 has been proposed to be the endonuclease responsible for mRNA cleavage. We created several Dom34 mutants and examined their effects on NGD in yeast. We identified mutations in several loops of the Dom34 structure that affect NGD. In contrast, mutations inactivating the proposed nuclease domain do not affect NGD in vivo. Moreover, we observed that overexpression of the Rps30a protein, a high copy suppressor of dom34Delta cold sensitivity, can restore some mRNA cleavage in a dom34Delta strain. These results identify important functional regions of Dom34 and suggest that the proposed endonuclease activity of Dom34 is not required for mRNA cleavage in NGD. We also provide evidence that the process of NGD is conserved in insect cells. On the basis of these results and the process of translation termination, we suggest a multistep model for the process of NGD.

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chainconjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.Tae2/Nemf | translational surveillance | protein quality control | cryo-EM | listerin/Ltn1 E3 ubiquitin ligase D uring the canonical termination and recycling steps of translation, stop codon recognition triggers factor-mediated hydrolysis of the nascent peptidyl-tRNA conjugate, nascent chain release, and ribosome splitting (1-3). Conversely, translation of aberrant mRNA, such as mRNA lacking stop codons ("nonstop mRNA"), renders 80S ribosomes stalled with nascent polypeptides (1-3). Furthermore, "nonstop proteins" cannot be corrected by quality control chaperones and have the potential to interfere with cellular function (3, 4). Not surprisingly, defective termination and recycling are under surveillance by a variety of mechanisms (1-3). In eukaryotes, "rescue factors" homologous to termination factors promote dissociation of translationally halted ribosomes in a stop codon-independent manner (5). However, because rescue factors lack peptidyl-tRNA hydrolase activity, their action results in nascent chains remaining stalled on the released 60S subunit.Ltn1 is the critical E3 ligase mediating ubiquitylation of aberrant proteins that become stalled on ribosomes during translation (4). Mutation of the Ltn1 mouse ortholog, listerin, causes neurodegeneration (6), suggesting an important function for this process. Ltn1 works together with several cofactors as part of the ribosome-associated quality control complex (RQC) (7-9) and appears to first associate with nascent chain-stalled 60S subunits together with two proteins of unknown function, Tae2 and Rqc1 (7, 9). Ltn1-mediated ubiquitylation of t...