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1. Visualizing the translation and packaging of HIV-1 full-length RNA

   https://doi.org/10.1073/pnas.1917590117  

   Chen, Jianbo; Liu, Yang; Wu, Bin; Nikolaitchik, Olga A.; Mohan, Preeti R.; Chen, Jiji; Pathak, Vinay K.; Hu, Wei-Shau (March 2020, Proceedings of the National Academy of Sciences)

   HIV-1 full-length RNA (HIV-1 RNA) plays a central role in viral replication, serving as a template for Gag/Gag-Pol translation and as a genome for the progeny virion. To gain a better understanding of the regulatory mechanisms of HIV-1 replication, we adapted a recently described system to visualize and track translation from individual HIV-1 RNA molecules in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Gag is an RNA binding protein that selects and packages HIV-1 RNA during virus assembly. A long-standing question in HIV-1 gene expression is whether Gag modulates HIV-1 RNA translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Using single-molecule tracking, we found that both translating and nontranslating RNAs exhibit dynamic cytoplasmic movement and can reach the plasma membrane, the major HIV-1 assembly site. However, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. These studies shed light on previously unknown aspects of HIV-1 gene expression and regulation. 

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2. Significant Differences in RNA Structure Destabilization by HIV-1 Gag∆p6 and NCp7 Proteins

   https://doi.org/10.3390/v12050484  

   McCauley, Micah J.; Rouzina, Ioulia; Li, Jasmine; Núñez, Megan E.; Williams, Mark C. (May 2020, Viruses)

   null (Ed.)

   Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag) polyprotein, binds the genomic RNA and facilitates packaging into new virions. It is not clear how the same protein, alone or as part of Gag, performs such different RNA binding functions in the viral life cycle. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. Comparing measured results with a model of discrete protein binding allows us to localize affected binding sites, in addition to quantifying hairpin stability. We find that, while both NCp7 and Gag∆p6 destabilize the TAR hairpin, Gag∆p6 binding is localized to two sites in the stem, while NCp7 targets sites near the top loop. Unlike Gag∆p6, NCp7 destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by ~104. Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion. 

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3. Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases

   https://doi.org/10.1021/acs.biochem.4c00271  

   Levintov, Lev; Gorai, Biswajit; Vashisth, Harish (October 2024, Biochemistry)

   The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt -shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signalinducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having a physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations. 

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4. Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

   https://doi.org/10.1038/s41467-023-38031-5  

   Wydorski, Pawel M.; Osipiuk, Jerzy; Lanham, Benjamin T.; Tesar, Christine; Endres, Michael; Engle, Elizabeth; Jedrzejczak, Robert; Mullapudi, Vishruth; Michalska, Karolina; Fidelis, Krzysztof; et al (December 2023, Nature Communications)

   Abstract The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub₂) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub₂combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub₂are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub₂modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function. 

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5. AID–RNA polymerase II transcription-dependent deamination of IgV DNA

   https://doi.org/10.1093/nar/gkz821  

   Pham, Phuong; Malik, Sohail; Mak, Chiho; Calabrese, Peter C; Roeder, Robert G; Goodman, Myron F (September 2019, Nucleic Acids Research)

   Abstract Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted. 

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