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Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag-PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag-PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]; however, previous studies suggested that HTLV-1 Gag-PM interactions and therefore virus assembly are less dependent on PI(4,5)P 2 . We have recently shown that PI(4,5)P 2 binds directly to HTLV-1 unmyristoylated MA [myr(-)MA] and that myr(-)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P 2 . Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA-membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P 2 -binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P 2 . Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Two non-covalently linked copies of the retrovirus genome are specifically recruited to the site of virus particle assembly and packaged into released particles. Retroviral RNA packaging requires RNA export of the unspliced genomic RNA from the nucleus, translocation of the genome to virus assembly sites, and specific interaction with Gag, the main viral structural protein. While some aspects of the RNA packaging process are understood, many others remain poorly understood. In this review, we provide an update on recent advancements in understanding the mechanism of RNA packaging for retroviruses that cause disease in humans, i.e., HIV-1, HIV-2, and HTLV-1, as well as advances in the understanding of the details of genomic RNA nuclear export, genome translocation to virus assembly sites, and genomic RNA dimerization.

Human immunodeficiency virus (HIV) mutagenesis is driven by a variety of internal and external sources, including the host APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypetide-like 3; A3) family of mutagenesis factors, which catalyze G-to-A transition mutations during virus replication. HIV-2 replication is characterized by a relative lack of G-to-A mutations, suggesting infrequent mutagenesis by A3 proteins. To date, the activity of the A3 repertoire against HIV-2 has remained largely uncharacterized, and the mutagenic activity of these proteins against HIV-2 remains to be elucidated. In this study, we provide the first comprehensive characterization of the restrictive capacity of A3 proteins against HIV-2 in cell culture using a dual fluorescent reporter HIV-2 vector virus. We found that A3F, A3G, and A3H restricted HIV-2 infectivity in the absence of Vif and were associated with significant increases in the frequency of viral mutants. These proteins increased the frequency of G-to-A mutations within the proviruses of infected cells as well. A3G and A3H also reduced HIV-2 infectivity via inhibition of reverse transcription and the accumulation of DNA products during replication. In contrast, A3D did not exhibit any restrictive activity against HIV-2, even at higher expression levels. Taken together, these results provide evidence that A3F, A3G, and A3H, but not A3D, are capable of HIV-2 restriction. Differences in A3-mediated restriction of HIV-1 and HIV-2 may serve to provide new insights in the observed mutation profiles of these viruses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Retroviral Gag targeting to the plasma membrane (PM) for assembly is mediated by the N-terminal matrix (MA) domain. For many retroviruses, Gag-PM interaction is dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). However, it has been shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P 2 than HIV-1, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to determine the structure of unmyristoylated MA (myr(-)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(-)MA binds to PI(4,5)P 2 via the polar head and that binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA-IP binding is governed by charge-charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using an NMR-based liposome binding assay, we show that PI(4,5)P 2 and phosphatidylserine enhance myr(-)MA binding in a synergistic fashion. Confocal microscopy data revealed formation of puncta on the PM of Gag expressing cells. However, G2A-Gag mutant, lacking myristoylation, is diffuse and cytoplasmic. These results suggest that although myr(-)MA binds to membranes, myristoylation appears to be key for formation of HTLV-1 Gag puncta on the PM. Altogether, these findings advance our understanding of a key mechanism in retroviral assembly., (Copyright © 2021 Elsevier Ltd. All rights reserved.)