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    Retroviral Genome Packaging

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    Retroviruses selectively package two copies of their RNA genomes from a cellular milieu that includes a substantial excess of host and non-genomic viral RNAs. Present understanding of the structural determinants and mechanism of retroviral genome packaging has been derived from combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical studies. Genetic experiments provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery.

    1. Introduction

    During virus assembly, all retroviruses package two copies of their 5ʹ-capped, unspliced RNA genomes, a requirement for strand transfer-mediate recombination during reverse transcription. Insights into the molecular structures and mechanisms responsible for genome packaging have been obtained by combinations of virological, molecular biological, and biophysical  methodologies. A method that has contributed considerably to our understanding of the structural determinants of packaging is solution-state nuclear magnetic resonance (NMR). A particularly attractive feature of NMR is its ability to probe the structures and dynamics of conformationally distinct biomolecule structures within a heterogeneous mixtures of equilibrating species. Disadvantages of the NMR approach include low proton density, poor chemical shift dispersion, and relatively low signal sensitivity and resolution that render studies of larger RNAs (> ~50 nucleotides) problematic. The development of new isotopic labeling methods and interplay of hybrid approaches has opened the door to surpassing the bounds of previously determined NMR structures. Here we summarize insights into the structures and mechanism of retroviral genome packaging that have been obtained by NMR. 

    2. Application of Nuclear Magnetic Resonance

    2.1. Protein Components Important for Genome Packaging

    Retroviral genome packaging and virus particle production are facilitated by Gag polyproteins, which consist of three structural domains (matrix; MA, capsid; CA, and nucleocapsid; NC) that are functionally important for retroviral replication: MA directs Gag to the plasma membrane for particle assembly, CA orchestrates the necessary protein-protein interactions required for Gag multimerization, and NC functions as an RNA binding domain for selective genome recognition. Each of these domains has been investigated using NMR either independently or in complex with their cognate lipid, protein, or nucleic acid partners. Following are some of the contributions to understanding retroviral proteins that have been advanced via NMR.

    2.2. Matrix

    NMR has been utilized in many instances to characterize the MA domain [1][2][3][4][5] in the context of its relationship to cytosolic trafficking [5][6][7][8][9], MA-RNA interactions [10], and MA-plasma membrane interactions. Structural elucidation of the MA domain consistently revealed a globular fold, of five ⍺-helices, a short 310 helical stretch, and a three-strand mixed β-sheet [11]. MA-plasma membrane NMR studies investigated a native co-translationally modified (myristoylation) protein revealing that it adopts both myristoyl exposed and sequestered conformations that do not perturb the tertiary fold [3]. Although NMR studies with water-soluble PI(4,5)P2 lipids containing truncated fatty acid chains showed that one of the acyl chains can bind to a hydrophobic cleft on MA (the so-called “extended lipid” binding mode) [12], later NMR studies with native PI(4,5)P2 molecules embedded in bicelles and liposomes supported models in which both fatty acid chains of PI(4,5)2 remain embedded in the liposome upon MA binding [6]. NMR was used to show that tRNALys3 interacts with residues in the basic patch region of MA, and that tRNALys3 binding inhibits MA interactions with PI(4,5)P2 enriched liposomes [10]

    2.3. Capsid

    The CA domain uses both its N- and C-terminal domains to participate in intermolecular contacts allowing CA multimers [13] and additional Gag-Gag interactions. NMR studies to investigate these contact interfaces were conducted with a construct comprising CACTD through NC (CACTD-SP1-NC) [14]. Data revealed that SP1 (spacer region 1) is conformationally labile and exists as unstructured (predominant) and helical (minor) states. There have also been studies on larger protein constructs spanning from the MA domain through NC (unmyristoylated MA-CA-SP1-NC, ~100 kDa); 1H-15N chemical shift perturbation mapping revealed structural changes that occur upon nucleic acid binding [15].

    2.4. Nucleocapsid

    NMR structures have been reported for isolated NC proteins from Human immunodeficiency virus (HIV-1/2), Mason-Pfizer monkey virus (MPMV), Moloney murine leukemia virus (MoMuLV), Mouse mammary tumor virus (MMTV), and Simian immunodeficiency virus (SIV). The proteins contain one or two copies of a CCHC-type zinc knuckle domain, and most studies indicate that they adopt structures that behave like “beads on a string” [16][17]. There are also several NMR structures for NC bound to viral RNA for different viruses such as HIV-1/2, MoMuLV, and Rous sarcoma virus (RSV). Each of which illustrates specific recognition of RNA ψ packaging elements that are facilitated by interactions with hydrophobic pockets of the NC zinc knuckles [18][19][20]. In addition to its role in genome packaging, the NC domain of Gag (or the mature NC protein) functions as an RNA chaperone to catalyze conformational rearrangements [21]. NMR studies showed that NC performs its chaperone activity by lowering the energy barrier required to break base pairs or by facilitating the formation of new base-pairs [22][21][23][24][25][26][27][28].

    2.5. RNA Components Important for Genome Packaging

    Retroviruses contain two copies of their RNA genomes [29], which functions in a number of replication processes from dimerization, packaging, transcriptional activation, splicing, and initiation of reverse transcription. Each of these processes are promoted by the elements located within the 5′-leader of the viral genome [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45], which is among the most conserved regions of  the ~9kb genome [46][47], ( A combination studies have identified minimal packaging regions within the 5′-leaders of HIV-1 [48], MoMuLV [49][50], and Rous Sarcoma Virus (RSV)[51][52][53][54][55][56], which are independently capable of directing heterologous RNAs into assembling virus-like particles (VLPs). These regions have been coined “core encapsidation signals” (ψCES) and are typically localized near residues that promote RNA dimerization [32][33][35][37]. Significant efforts have been aimed toward elucidating the three-dimensional structures of the ψCES regions and other functional elements within the 5ʹ-leader in order to better understand their mechanism of action in relationship to genome packaging. Summarized below are few of the NMR studies that have been instrumental in the advancement of the not only the field of retroviruses but also RNA structural biology.

    2.6. HIV-1 (TAR)

    The trans-activation region (TAR) is a 59-nt sequence located at the 5′-end of the 5ʹ-leader and is essential in Tat-mediated transcriptional activation [57][58][59][60][61]. The first NMR  structures for TAR and TAR-Tat complexes revealed the structural elements that are critical for Tat binding specifically bulge residue (U23), two base-pairs immediately downstream from the bulge (G26-C39 and A27-U38), and three phosphate groups [62][63]. Continued NMR studies revealed additional unique characteristics of TAR including, a base-triple that forms between U23 and A27-U38, which functions in stabilizing the interactions between Tat arginine residue G26 with phosphates in the major grooves of the RNA [62][63].

    2.7. HIV-1 (ψCES)

    Structural studies of ΨCES were facilitated by implementing mutations that reduced  the size of the RNA without altering the remaining base pairing or function. At 155 nt, this construct was five times larger than the average size of RNA structures previously solved by NMR at the time [64]. Complications with signal resolution for an RNA this size were still not completely resolved with the traditional methods of nucleotide-specific 2H-labeling. However, a novel approach involving the non-covalent annealing of differentially 2H-labeled RNA fragments aided in the necessary resolution for the identification of a unique tandem three-way junction formation for the RNA [64].

    2.8. HIV-1 (Intact 5ʹ-leader)

    It was surprising that the dimeric form of the intact 5′-leader of the NL4-3 strain of HIV-1 (molecular weight of ~230 kDa) produced interpretable NMR data [65]. The quality of the spectra revealed that the RNA is comprised of independently folded subdomains. This in combination with 2H-labeling enabled for the direct detection of predicted secondary structural elements  [48][65][66]. Other studies of the intact leader utilized 2H-edited to serve in the development of a  new diagnostic tool, called long-range Adenosine Interaction Detection (lrAID) [65], which takes advantage of a [AAU]:[AUU] base pairing sequence that produces resolved adenosine signals even in larger RNA constructs.

    2.9. HIV-1 (5ʹ-Transcriptional Start Site Heterogeneity and Capping)

    The promoter of the HIV-1 proviral DNA contains three sequential guanosines that can function as the start site for transcription. In cells, RNAs are transcribed by RNA Polymerase II and are co-transcriptionally capped with 5ʹ-5ʹ triphosphate linked 7-methylguanosine (7MeG) [67][68][69][70]. In vitro, capped RNAs containing a single 5ʹ-guanosine preferentially form dimers whereas those that begin with two or three 5ʹ-guanosines preferentially form monomers [71]. NMR studies revealed that the capped 1G leader adopts a structure in which the cap is sandwiched between the TAR and polyA helices, which are co-linearly stacked in an “end-to-end” manner [72]. 2H-edited NMR studies of the capped 2G/3G leader RNAs, revealed extensive structural remodeling compared to the capped 1G RNA [72] and that the cap residue is exposed and disordered [72]. These studies provided a structural explanation for how transcriptional start heterogeneity modulates the structure, function, and fate of HIV-1 RNA.

    3. Conclusion

    The above studies illustrate the versatility of NMR and its utility for developing detailed understanding of retroviral structural biology. Future studies of genome packaging will likely focus on even larger RNAs and protein-RNA complexes. Some steps have been made to enable NMR studies of larger RNAs, including the development of site-specific isotopic labeling [73] and the expanded use of 1H-15N correlated NMR methods [74]. But as questions shift to larger systems, new approaches involving hybrid methodologies, advancements in labeling techniques, and solid-state NMR, will likely make important future contributions.

    The entry is from 10.3390/v12101115


    1. Matthews, S.; Barlow, P.; Boyd, J.; Barton, G.; Russell, R.; Mills, H.; Cunningham, M.; Meyers, N.; Burns, N.; Clark, N.; et al. Structural similarity between the p17 matrix protein of HIV-1 and interferon-g. Nature 1994, 370, 666–668.
    2. Tang, C.; Ndassa, Y.; Summers, M.F. Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein. Nat. Struct. Biol. 2002, 9, 537–543.
    3. Tang, C.; Loeliger, E.; Luncsford, P.; Kinde, I.; Beckett, D.; Summers, M.F. Entropic switch regulates myristate exposure in the HIV-1 matrix protein. Proc. Natl. Acad. Sci. USA 2004, 101, 517–522.
    4. Saad, J.S.; Loeliger, E.; Luncsford, P.; Liriano, M.; Tai, J.; Kim, A.; Miller, J.; Joshi, A.; Freed, E.O.; Summers, M.F. Point mutations in the HIV-1 matrix protein turn off the myristyl switch. J. Mol. Biol. 2007, 366, 574–585.
    5. Saad, J.S.; Ablan, S.D.; Ghanam, R.H.; Kim, A.; Andrews, K.; Nagashima, K.; Soheilian, F.; Freed, E.O.; Summers, M.F. Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting. J. Mol. Biol. 2008, 382, 434–447.
    6. Mercredi, P.Y.; Bucca, N.; Loeliger, B.; Gaines, C.R.; Mehta, M.; Bhargava, P.; Tedbury, P.R.; Charlier, L.; Floquet, N.; Muriaux, D.; et al. Structural and molecular determinants of membrane binding by the HIV-1 matrix protein. J. Mol. Biol. 2016, 428, 1637–1655.
    7. Saad, J.S.; Miller, J.; Tai, J.; Kim, A.; Ghanam, R.H.; Summers, M.F. Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly. Proc. Natl. Acad. Sci. USA 2006, 103, 11364–11369.
    8. Saad, J.S.; Kim, A.; Ghanam, R.H.; Dalton, A.K.; Vogt, M.V.; Wu, Z.; Lu, W.; Summers, M.F. Mutations that mimic phosphorylation of the HIV-1 matrix protein: Implications for trafficking. Protein Sci. 2007, 16, 1793–1797.
    9. Murphy, R.E.; Smal, A.B.; Blach, J.; Mas, V.; Prevelige, P.E.; Saad, J.S. Structural and biophysical characterizations of HIV-1 matrix trimer binding to lipid nanodiscs shed light on virus assembly. J. Biol. Chem. 2019, 294, 18600–18612.
    10. Gaines, C.R.; Tkacik, E.; Rivera-Oven, A.; Somani, P.; Achimovich, A.; Alabi, T.; Zhu, A.; Getachew, N.; Yang, A.L.; McDonough, M.; et al. HIV-1 Matrix protein interactions with tRNA: Implications for membrane targeting. J. Mol. Biol. 2018, 430, 2113–2127.
    11. Massiah, M.A.; Starich, M.R.; Paschall, C.; Summers, M.F.; Christensen, A.M.; Sundquist, W.I. Three dimensional structure of the human immunodeficiency virus type 1 matrix protein. J. Mol. Biol. 1994, 244, 198–223.
    12. Saad,J.S.;Miller,J.;Tai,J.;Kim,A.;Ghanam,R.H.;Summers,M.F.Structural basis for targeting HIV-1Gag proteins to the plasma membrane for virus assembly. Proc. Natl. Acad. Sci. USA 2006, 103, 11364–11369.
    13. Ganser-Pornillos, B.K.; Yeager, M.; Sundquist, W.I. The structural biology of HIV assembly. Curr. Opin. Struct. Biol. 2008, 18, 203–217.
    14. Newman, J.L.; Butcher, E.W.; Patel, D.T.; Mikhaylenko, Y.; Summers, M.F. Flexibility in the P2 domain of the HIV-1 Gag polyprotein. Protein Sci. 2004, 13, 2101–2107.
    15. Deshmukh, L.; Ghirlando, R.; Clore, G.M. Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy. Proc. Natl. Acad. Sci. USA 2015, 112, 3374–3379.
    16. South, T.L.; Blake, P.R.; Sowder, R.C., 3rd.; Arthur, L.O.; Henderson, L.E.; Summers, M.F. The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers. Biochemistry 1990, 29, 7786–7789.
    17. Summers, M.F.; Henderson, L.E.; Chance, M.R.; Bess, J.W.J.; South, T.L.; Blake, P.R.; Sagi, I.; Perez-Alvarado, G.; Sowder, R.C.I.; Hare, D.R.; et al. Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1. Protein Sci. 1992, 1, 563–574.
    18. D’Souza, V.; Summers, M.F. Structural basis for packaging the dimeric genome of Moloney Murine Leukaemia Virus. Nature 2004, 431, 586–590.
    19. Zhou, J.; Bean, R.L.; Vogt, V.M.; Summers, M.F. Solution structure of the Rous sarcoma virus nucleocapsid protein:uY RNA packaging signal complex. J. Mol. Biol. 2007, 365, 453–467.
    20. De Guzman, R.N.; Wu, Z.R.; Stalling, C.C.; Pappalardo, L.; Borer, P.N.; Summers, M.F. Structure of the HIV-1 nucleocapsid protein bound to the SL3 Y-RNA recognition element. Science 1998, 279, 384–388.
    21. Levin, J.G.; Guo, J.; Rouzina, I.; Musier-Forsyth, K. Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: Critical role in reverse transcription and molecular mechanism. Prog. Nucleic Acid Res. Mol. Biol. 2005, 80, 217–286.
    22. Sleiman, D.; Goldschmidt, V.; Barraud, P.; Marquet, R.; Paillart, J.C.; Tisne, C. Initiation of HIV-1 reverse transcription and functional role of nucleocapsid-mediated tRNA/viral genome interactions. Virus Res. 2012, 169, 324–339.
    23. Johnson, P.E.; Turner, R.B.; Wu, Z.-R.; Hairston, L.; Guo, J.; Levin, J.G.; Summers, M.F. A mechanism for (+) strand transfer enhancement by the HIV-1 nucleocapsid protein during reverse transcription. Biochemistry 2000, 39, 9084–9091.
    24. Barraud, P.; Gaudin, C.; Dardel, F.; Tisne, C. New insights into the formation of HIV-1 reverse transcription initiation complex. Biochimie 2007, 89, 1204–1210.
    25. Tisne, C.; Roques, B.P.; Dardel, F. Heteronuclear NMR studies of the interaction of tRNA(Lys)3 with HIV-1 nucleocapsid protein. J. Mol. Biol. 2001, 306, 443–454.
    26. Bourbigot, S.; Ramalanjaona, N.; Boudier, C.; Salgado, G.F.; Roques, B.P.; Mely, Y.; Bouaziz, S.; Morellet, N. How the HIV-1 nucleocapsid protein binds and destabilises the (-)primer binding site during reverse transcription. J. Mol. Biol. 2008, 383, 1112–1128.
    27. Bazzi, A.; Zargarian, L.; Chaminade, F.; Boudier, C.; de Rocquigny, H.; Rene, B.; Mely, Y.; Fosse, P.; Mauffret, O. Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: Role of sugar deoxyriboses in the binding polarity of NC. Nucleic Acids Res. 2011, 39, 3903–3916.
    28. Belfetmi, A.; Zargarian, L.; Tisne, C.; Sleiman, D.; Morellet, N.; Lescop, E.; Maskri, O.; Rene, B.; Mely, Y.; Fosse, P.; et al. Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein. RNA 2016, 22, 506–517.
    29. Vogt, V.M. Retroviral virions and genomes. In Retroviruses; Coffin, J.M., Hughes, S.H., Varmus, H.E., Eds.; Cold Spring Harbor Laboratory Press: Plainview, NY, USA, 1997; Volume 1, pp. 27–69.
    30. Berkowitz, R.; Fisher, J.; Goff, S.P. RNA packaging. Curr. Top. Microbiol. Immun. 1996, 214, 177–218.
    31. Rein, A. Retroviral RNA packaging: A review. Arch. Virol. 1994, 9, 513–522.
    32. Greatorex, J.; Lever, A. Retroviral RNA dimer linkage. J. Gen. Virol. 1998, 79, 2877–2882.
    33. Paillart, J.-C.; Marquet, R.; Skripkin, E.; Ehresmann, C.; Ehresmann, B. Dimerization of retroviral genomic RNAs: Structural and functional implications. Biochimie 1996, 78, 639–653.
    34. Jewell, N.A.; Mansky, L.M. In the beginning: Genome recognition, RNA encapsidation and the initiation of complex retrovirus assembly. J. Gen. Virol. 2000, 81, 1889–1899.
    35. Paillart, J.-C.; Shehu-Xhilaga, M.; Marquet, R.; Mak, J. Dimerization of retroviral RNA genomes: An inseparable pair. Nat. Rev. Microbiol. 2004, 2, 461–472.
    36. Russell, R.S.; Liang, C.; Wainberg, M.A. Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably? Retrovirology 2004, 1, 23.
    37. Greatorex, J. The retroviral RNA dimer linkage: Different structures may reflect different roles. Retrovirology 2004, 1, 22.
    38. D’Souza, V.; Summers, M.F. How retroviruses select their genomes. Nat. Rev. Microbiol. 2005, 3, 643–655.
    39. Johnson, S.F.; Telesnitsky, A. Retroviral RNA dimerization and packaging: The what, how, when, where, and why. PLoS Pathog. 2010, 6.
    40. Kuzembayeva, M.; Dilley, K.; Sardo, L.; Hu, W.-S. Life of psi: How full-length HIV-1 RNAs become packaged genomes in the viral particles. Virology 2014, 454, 362–370.
    41. Lu, K.; Heng, X.; Summers, M.F. Structural determinants and mechanism of HIV-1 genome packaging. J. Mol. Biol. 2011, 410, 609–633.
    42. Hellmund, C.; Lever, A.M. Coordination of genomic RNA packaging with viral assembly in HIV-1. Viruses 2016, 8, 192.
    43. Mailler, E.; Bernacchi, S.; Marquet, R.; Paillart, J.C.; Vivet-Boudou, V.; Smyth, R.P. The life-cycle of the HIV-1 Gag-RNA complex. Viruses 2016, 8, 248.
    44. Bieniasz, P.; Telesnitsky, A. Multiple, Switchable protein: RNA interactions regulate human immunodeficiency virus type 1 assembly. Annu. Rev. Virol. 2018, 5, 165–183.
    45. Rein, A. RNA Packaging in HIV. Trends Microbiol. 2019, 27, 715–723.
    46. Coffin, J.M.; Hughes, S.H.; Varmus, H.E. Retroviruses; Cold Spring Harbor Laboratory Press: Plainview, NY, USA, 1997.
    47. Lever, A.M. HIV-1 RNA packaging. Adv. Pharmacol. 2007, 55, 1–32.
    48. Heng, X.; Kharytonchyk, S.; Garcia, E.L.; Lu, K.; Divakaruni, S.S.; LaCotti, C.; Edme, K.; Telesnitsky, A.; Summers, M.F. Identification of a minimal region of the HIV-1 5’-leader required for RNA dimerization, NC binding, and packaging. J. Mol. Biol. 2012, 417, 224–239.
    49. Adam, M.A.; Miller, A.D. Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions. J. Virol. 1988, 62, 3802–3806.
    50. Mougel, M.; Barklis, E. A role for two hairpin structures as a core RNA encapsidation signal in murine leukemia virus virions. J. Virol. 1997, 71, 8061–8065.
    51. Aronoff, R.; Linial, M. Specificity of retroviral RNA packaging. J. Virol. 1991, 65, 71–80.
    52. Aronoff, R.; Hajjar, A.M.; Linial, M.L. Avian retroviral RNA encapsidation: Reexamination of functional 5’ RNA sequences and the role of nucleocapsid Cys-His motifs. J. Virol. 1993, 67, 178–188.
    53. Banks, J.D.; Yeo, A.; Green, K.; Cepeda, F.; Linial, M.L. A minimal avian retroviral packaging sequence has a complex structure. J. Virol. 1998, 72, 6190–6194.
    54. Doria-Rose, N.A.; Vogt, V.M. In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal. J. Virol. 1998, 72, 8073–8082.
    55. Banks, J.D.; Kealoha, B.O.; Linial, M.L. An MY containing heterologous RNA, but not env mRNA, is efficiently packaged into avian retroviral particles. J. Virol. 1999, 73, 8926–8933.
    56. Banks, J.D.; Linial, M.L. Secondary structure analysis of a minimal avian leukosis-sarcoma virus packaging signal. J. Virol. 2000, 74, 456–464.
    57. Wimmer, J.; Fujinaga, K.; Taube, R.; Cujec, T.P.; Zhu, Y.; Peng, J.; Price, D.H.; Peterlin, B.M. Interactions between Tat and TAR and human immunodeficiency virus replication are facilitated by human cyclin T1 but not cyclins T2a or T2b. Virology 1999, 255, 182–189.
    58. Brady, J.; Kashanchi, F. Tat gets the “green” light on transcription initiation. Retrovirology 2005, 2, 69.
    59. Laspia, M.F.; Rice, A.P.; Mathews, M.B. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell 1989, 59, 283–292.
    60. Chulze-Gahmen, U.; Hurley, J.H. Structural mechanism for HIV-1 TAR loop recognition by Tat and the super elongation complex. Proc. Natl. Acad. Sci. USA 2018, 115, 12973–12978.
    61. Chavali, S.S.; Bonn-Breach, R.; Wedekind, J.E. Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery. J. Biol. Chem. 2019, 294, 9326–9341.
    62. Puglisi, J.D.; Tan, R.; Calnan, B.J.; Frankel, A.D.; Williamson, J.R. Conformation of the TAR RNA-arginine complex by NMR spectroscopy. Science 1992, 257, 76–80.
    63. Puglisi, J.D.; Chen, L.; Frankel, A.D.; Williamson, J.R. Role of RNA structure in arginine recognition of TAR RNA. Proc. Natl. Acad. Sci. USA 1993, 90, 3680–3684.
    64. Keane, S.C.; Heng, X.; Lu, K.; Kharytonchyk, S.; Ramakrishnan, V.; Carter, G.; Barton, S.; Hosic, A.; Florwick, A.; Santos, J.; et al. Structure of the HIV-1 RNA packaging signal. Science 2015, 348, 917–921.
    65. Lu, K.; Heng, X.; Garyu, L.; Monti, S.; Garcia, E.; Kharytonchyk, S.; Dorjsuren, B.; Kulandaivel, G.; Jones, S.; Hiremath, A.; et al. NMR detection of structures in the HIV-1 5´-leader RNA that regulate genome packaging. Science 2011, 344, 242–245.
    66. Keane, S.C.; Van, V.; Frank, H.M.; Sciandra, C.A.; McCowin, S.; Santos, J.; Heng, X.; Summers, M.F. NMR detection of intermolecular interaction sites in the dimeric 5’-leader of the HIV-1 genome. Proc. Natl. Acad. Sci. USA 2016, 113, 13033–13038.
    67. Ya-Lin Chiu; Elizabeth Coronel; C. Kiong Ho; Stewart Shuman; Tariq M. Rana; HIV-1 Tat Protein Interacts with Mammalian Capping Enzyme and Stimulates Capping of TAR RNA. Journal of Biological Chemistry 2001, 276, 12959-12966, 10.1074/jbc.m007901200.
    68. Meisheng Zhou; Longwen Deng; Fatah Kashanchi; John N. Brady; Aaron J. Shatkin; Ajit Kumar; The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA. Proceedings of the National Academy of Sciences 2003, 100, 12666-12671, 10.1073/pnas.1835726100.
    69. Thomas M. Menees; Barbara Müller; Hans-Georg Kräusslich; The Major 5′ End of HIV Type 1 RNA Corresponds to G456. AIDS Research and Human Retroviruses 2007, 23, 1042-1048, 10.1089/aid.2006.0275.
    70. Amit Sharma; Alper Yilmaz; Kim Marsh; Alan Cochrane; K Boris-Lawrie; Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity. PLoS Pathogens 2012, 8, e1002612, 10.1371/journal.ppat.1002612.
    71. Siarhei Kharytonchyk; Sarah Monti; Philip J. Smaldino; Verna Van; Nicholas C. Bolden; Joshua D. Brown; Emily Russo; Canessa Swanson; Alex Shuey; Alice Telesnitsky; et al. Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome. Proceedings of the National Academy of Sciences 2016, 113, 13378-13383, 10.1073/pnas.1616627113.
    72. Joshua D. Brown; Siarhei Kharytonchyk; Issac Chaudry; Aishwarya S. Iyer; Hannah Carter; Ghazal Becker; Yash Desai; Lindsay Glang; Seung H. Choi; Karndeep Singh; et al. Structural basis for transcriptional start site control of HIV-1 RNA fate.. null 2020, 368, 413-417, .
    73. Liu,Y.;Holmstrom,E.;Zhang,J.;Yu,P.;Wang,J.;Dyba,M.A.;Chen,D.;Ying,J.;Lockett,S.;Nesbitt,D.J.; et al. Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature 2015, 522, 368–372.
    74. Marchant, J.; Bax, A.; Summers, M.F. Accurate measurement of residual dipolar couplings in large RNAs by Variable flip angle NMR. J. Am. Chem. Soc. 2018, 140, 6978–6983, doi:10.1021/jacs.8b03298.