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• W2038245782 abstract "The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication. The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication. The untranslated 5′ leader region (Fig. 1A) is the most conserved part of the HIV-1 RNA genome when comparing the nucleotide sequence of different virus isolates. The leader can fold multiple stem-loop structures that regulate various steps of the viral replication cycle, including the poorly understood processes of RNA dimerization and RNA packaging in virion particles. The DIS 3The abbreviations used are: DISdimerization initiation signalKLkissing loopEDextended dimerLDIlong distance interactionntnucleotideBMHbranched multiple-hairpins. hairpin folds a stable stem-loop structure that is sufficient to produce HIV-1 RNA dimers in vitro (1.Darlix J.L. Gabus C. Nugeyre M.T. Clavel F. Barré-Sinoussi F. cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.J. Mol. Biol. 1990; 216: 689-699Crossref PubMed Scopus (308) Google Scholar, 2.Marquet R. Baudin F. Gabus C. Darlix J.L. Mougel M. Ehresmann C. Ehresmann B. Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.Nucleic Acids Res. 1991; 19: 2349-2357Crossref PubMed Scopus (159) Google Scholar, 3.Paillart J.C. Marquet R. Skripkin E. Ehresmann B. Ehresmann C. Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA.J. Biol. Chem. 1994; 269: 27486-27493Abstract Full Text PDF PubMed Google Scholar, 4.Paillart J.C. Skripkin E. Ehresmann B. Ehresmann C. Marquet R. A loop-loop “kissing” complex is the essential part of the dimer linkage of genomic HIV-1 RNA.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5572-5577Crossref PubMed Scopus (233) Google Scholar, 5.Skripkin E. Paillart J.C. Marquet R. Ehresmann B. Ehresmann C. Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 4945-4949Crossref PubMed Scopus (370) Google Scholar). It harbors a palindrome sequence in the loop (GCGCGC for HIV-1 subtype B), such that two DIS hairpins can engage in kissing loop (KL) base pairing interaction (Fig. 1, B and C), thus forming “loose” RNA dimers (6.Berkhout B. van Wamel J.L. Role of the DIS hairpin in replication of human immunodeficiency virus type 1.J. Virol. 1996; 70: 6723-6732Crossref PubMed Google Scholar, 7.Ennifar E. Walter P. Ehresmann B. Ehresmann C. Dumas P. Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site.Nat. Struct. Biol. 2001; 8: 1064-1068Crossref PubMed Scopus (174) Google Scholar, 8.St Louis D.C. Gotte D. Sanders-Buell E. Ritchey D.W. Salminen M.O. Carr J.K. McCutchan F.E. Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection in vitro.J. Virol. 1998; 72: 3991-3998Crossref PubMed Google Scholar). Heat treatment or incubation with the viral nucleocapsid protein triggers opening of the hairpin stem to allow extended interstrand base pairing and formation of a more stable extended dimer (ED) (Fig. 1, B and C) (9.Feng Y.X. Copeland T.D. Henderson L.E. Gorelick R.J. Bosche W.J. Levin J.G. Rein A. HIV-1 nucleocapsid protein induces “maturation” of dimeric retroviral RNA in vitro.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 7577-7581Crossref PubMed Scopus (155) Google Scholar, 10.Muriaux D. Fossé P. Paoletti J. A kissing complex together with a stable dimer is involved in the HIV-1Lai RNA dimerization process in vitro.Biochemistry. 1996; 35: 5075-5082Crossref PubMed Scopus (141) Google Scholar, 11.Theilleux-Delalande V. Girard F. Huynh-Dinh T. Lancelot G. Paoletti J. The HIV-1(Lai) RNA dimerization: thermodynamic parameters associated with the transition from the kissing complex to the extended dimer.Eur. J. Biochem. 2000; 267: 2711-2719Crossref PubMed Scopus (35) Google Scholar). Such ED RNA forms have only been described in vitro, and their in vivo relevance remains unclear. dimerization initiation signal kissing loop extended dimer long distance interaction nucleotide branched multiple-hairpins. We previously reported that structural rearrangements in the leader regulate RNA dimerization in vitro (12.Abbink T.E. Berkhout B. A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon.J. Biol. Chem. 2003; 278: 11601-11611Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 13.Abbink T.E. Ooms M. Haasnoot P.C. Berkhout B. The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation.Biochemistry. 2005; 44: 9058-9066Crossref PubMed Scopus (92) Google Scholar, 14.Huthoff H. Berkhout B. Multiple secondary structure rearrangements during HIV-1 RNA dimerization.Biochemistry. 2002; 41: 10439-10445Crossref PubMed Scopus (73) Google Scholar). In vitro RNA dimerization of HIV-1 transcripts was prevented by formation of the so-called long distance interaction (LDI) conformation in which the DIS sequences are engaged in an intramolecular, long distance base pairing interaction with the upstream poly(A) hairpin (Fig. 1). As a result, the DIS palindrome is not available for initiating RNA dimer formation. Alternative HIV-1 RNA folding schemes have recently been proposed (15.Lu K. Heng X. Garyu L. Monti S. Garcia E.L. Kharytonchyk S. Dorjsuren B. Kulandaivel G. Jones S. Hiremath A. Divakaruni S.S. LaCotti C. Barton S. Tummillo D. Hosic A. Edme K. Albrecht S. Telesnitsky A. Summers M.F. NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging.Science. 2011; 334: 242-245Crossref PubMed Scopus (194) Google Scholar, 16.Sakuragi J. Ode H. Sakuragi S. Shioda T. Sato H. A proposal for a new HIV-1 DLS structural model.Nucleic Acids Res. 2012; 40: 5012-5022Crossref PubMed Scopus (22) Google Scholar, 17.Watts J.M. Dang K.K. Gorelick R.J. Leonard C.W. Bess Jr., J.W. Swanstrom R. Burch C.L. Weeks K.M. Architecture and secondary structure of an entire HIV-1 RNA genome.Nature. 2009; 460: 711-716Crossref PubMed Scopus (598) Google Scholar), but most models converge on the concept of regulation of RNA dimerization through RNA conformational changes, with one of the conformations being dimerization-incompetent because of the absence of the DIS hairpin or shielding of the DIS palindrome. This inhibition of RNA dimerization could be relieved by heat treatment or incubation with the HIV-1 nucleocapsid chaperone protein (9.Feng Y.X. Copeland T.D. Henderson L.E. Gorelick R.J. Bosche W.J. Levin J.G. Rein A. HIV-1 nucleocapsid protein induces “maturation” of dimeric retroviral RNA in vitro.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 7577-7581Crossref PubMed Scopus (155) Google Scholar, 18.Huthoff H. Berkhout B. Two alternating structures of the HIV-1 leader RNA.RNA. 2001; 7: 143-157Crossref PubMed Scopus (179) Google Scholar). Both treatments trigger refolding of the LDI conformation into the dimerization-competent BMH (branched multiple hairpin) structure, which exposes the DIS hairpin (Fig. 1B). Infectious HIV-1 particles contain two genomic RNA molecules that are noncovalently linked near their 5′ end, possibly at more than one position (19.Höglund S. Ohagen A. Goncalves J. Panganiban A.T. Gabuzda D. Ultrastructure of HIV-1 genomic RNA.Virology. 1997; 233: 271-279Crossref PubMed Scopus (85) Google Scholar). It is thought that the native dimer structure of the RNA genome is important for its efficient reverse transcription into DNA, a complex process that includes two strand transfer events (20.van Wamel J.L. Berkhout B. The first strand transfer during HIV-1 reverse transcription can occur either intramolecularly or intermolecularly.Virology. 1998; 244: 245-251Crossref PubMed Scopus (48) Google Scholar). RNA genome dimers are also thought to facilitate recombination during reverse transcription, which contributes to the generation of HIV genetic diversity (21.Moore M.D. Hu W.S. HIV-1 RNA dimerization: it takes two to tango.AIDS Rev. 2009; 11: 91-102PubMed Google Scholar, 22.Nikolaitchik O.A. Galli A. Moore M.D. Pathak V.K. Hu W.S. Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay.J. Mol. Biol. 2011; 407: 521-531Crossref PubMed Scopus (17) Google Scholar). RNA recombination rates are indeed significantly reduced when the DIS palindrome sequence is altered (22.Nikolaitchik O.A. Galli A. Moore M.D. Pathak V.K. Hu W.S. Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay.J. Mol. Biol. 2011; 407: 521-531Crossref PubMed Scopus (17) Google Scholar, 23.Chen J. Nikolaitchik O. Singh J. Wright A. Bencsics C.E. Coffin J.M. Ni N. Lockett S. Pathak V.K. Hu W.S. High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13535-13540Crossref PubMed Scopus (156) Google Scholar, 24.Moore M.D. Fu W. Nikolaitchik O. Chen J. Ptak R.G. Hu W.S. Dimer initiation signal of human immunodeficiency virus type 1: its role in partner selection during RNA copackaging and its effects on recombination.J. Virol. 2007; 81: 4002-4011Crossref PubMed Scopus (80) Google Scholar). Studies on the conformation of genomic RNA inside virus particles are intrinsically difficult. Early electron microscopy studies indicated multiple RNA-RNA contacts along the dimeric genome, with the most stable interaction located near the 5′ ends (25.Bender W. Davidson N. Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules.Cell. 1976; 7: 595-607Abstract Full Text PDF PubMed Scopus (158) Google Scholar). Stabilization of the RNA dimer was reported during maturation of newly assembled virion particles, but details on the molecular events remained obscure (26.Fu W. Rein A. Maturation of dimeric viral RNA of Moloney murine leukemia virus.J. Virol. 1993; 67: 5443-5449Crossref PubMed Google Scholar, 27.Song R. Kafaie J. Yang L. Laughrea M. HIV-1 viral RNA is selected in the form of monomers that dimerize in a three-step protease-dependent process; the DIS of stem-loop 1 initiates viral RNA dimerization.J. Mol. Biol. 2007; 371: 1084-1098Crossref PubMed Scopus (47) Google Scholar). The HIV-1 DIS motif is considered critical for RNA dimerization (3.Paillart J.C. Marquet R. Skripkin E. Ehresmann B. Ehresmann C. Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA.J. Biol. Chem. 1994; 269: 27486-27493Abstract Full Text PDF PubMed Google Scholar, 5.Skripkin E. Paillart J.C. Marquet R. Ehresmann B. Ehresmann C. Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 4945-4949Crossref PubMed Scopus (370) Google Scholar, 28.Laughrea M. Jetté L. A 19-nucleotide sequence upstream of the 5′ major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA.Biochemistry. 1994; 33: 13464-13474Crossref PubMed Scopus (223) Google Scholar, 29.Laughrea M. Jetté L. Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248–271 are dispensable for dimer formation.Biochemistry. 1996; 35: 1589-1598Crossref PubMed Scopus (173) Google Scholar), but it may also regulate RNA packaging (30.Nikolaitchik O.A. Dilley K.A. Fu W. Gorelick R.J. Tai S.H. Soheilian F. Ptak R.G. Nagashima K. Pathak V.K. Hu W.S. Dimeric RNA recognition regulates HIV-1 genome packaging.PLoS Pathog. 2013; 9: e1003249Crossref PubMed Scopus (64) Google Scholar). Surprisingly, some studies suggested that the DIS palindrome is dispensable for RNA dimerization in vivo: its deletion or alteration to a smaller palindromic sequence does not affect RNA dimer stability inside virus particles, although RNA packaging was reduced (6.Berkhout B. van Wamel J.L. Role of the DIS hairpin in replication of human immunodeficiency virus type 1.J. Virol. 1996; 70: 6723-6732Crossref PubMed Google Scholar). In addition, cell type-specific DIS defects were reported (31.Hill M.K. Shehu-Xhilaga M. Campbell S.M. Poumbourios P. Crowe S.M. Mak J. The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells.J. Virol. 2003; 77: 8329-8335Crossref PubMed Scopus (56) Google Scholar, 32.Jones K.L. Sonza S. Mak J. Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription.Nucleic Acids Res. 2008; 36: 1578-1588Crossref PubMed Scopus (20) Google Scholar). Additional RNA contact sites have been proposed for the upstream HIV-1 TAR hairpin and other leader motifs, including the SL3 region downstream (33.Andersen E.S. Contera S.A. Knudsen B. Damgaard C.K. Besenbacher F. Kjems J. Role of the trans-activation response element in dimerization of HIV-1 RNA.J. Biol. Chem. 2004; 279: 22243-22249Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 34.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: 224-239Crossref PubMed Scopus (78) Google Scholar, 35.Huthoff H. Berkhout B. Mutations in the TAR hairpin affect the equilibrium between alternative conformations of the HIV-1 leader RNA.Nucleic Acids Res. 2001; 29: 2594-2600Crossref PubMed Scopus (38) Google Scholar, 36.Song R. Kafaie J. Laughrea M. Role of the 5′ TAR stem-loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA.Biochemistry. 2008; 47: 3283-3293Crossref PubMed Scopus (41) Google Scholar, 37.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 psi-RNA recognition element.Science. 1998; 279: 384-388Crossref PubMed Scopus (609) Google Scholar). However, such studies require a careful analysis because leader mutations may induce RNA misfolding and conformational changes that indirectly trigger or impede RNA dimerization (38.Das A.T. Vrolijk M.M. Harwig A. Berkhout B. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging.Retrovirology. 2012; 9: 59Crossref PubMed Scopus (22) Google Scholar, 39.Vrolijk M.M. Ooms M. Harwig A. Das A.T. Berkhout B. Destabilization of the TAR hairpin affects the structure and function of the HIV-1 leader RNA.Nucleic Acids Res. 2008; 36: 4352-4363Crossref PubMed Scopus (29) Google Scholar). Full-length HIV-1 RNA genomes are selected from an excess of cellular and subgenomic viral RNA during the virus assembly process and encapsidated by the Gag proteins. RNA dimerization and packaging are tightly linked processes: if proper RNA dimerization is compromised, the virus particles contain less genomic RNA and are less infectious (6.Berkhout B. van Wamel J.L. Role of the DIS hairpin in replication of human immunodeficiency virus type 1.J. Virol. 1996; 70: 6723-6732Crossref PubMed Google Scholar, 40.Ohishi M. Nakano T. Sakuragi S. Shioda T. Sano K. Sakuragi J. The relationship between HIV-1 genome RNA dimerization, virion maturation and infectivity.Nucleic Acids Res. 2011; 39: 3404-3417Crossref PubMed Scopus (29) Google Scholar, 41.Russell R.S. Liang C. Wainberg M.A. Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?.Retrovirology. 2004; 1: 23Crossref PubMed Scopus (102) Google Scholar, 42.Sakuragi J. Ueda S. Iwamoto A. Shioda T. Possible role of dimerization in human immunodeficiency virus type 1 genome RNA packaging.J. Virol. 2003; 77: 4060-4069Crossref PubMed Scopus (58) Google Scholar). The initial RNA-RNA dimer is formed in the virus-producing cell and is subsequently packaged. During virus particle maturation the stability of the HIV-1 RNA dimer increases, which depends on viral Protease activity and processing of the Gag-Pol polyprotein (27.Song R. Kafaie J. Yang L. Laughrea M. HIV-1 viral RNA is selected in the form of monomers that dimerize in a three-step protease-dependent process; the DIS of stem-loop 1 initiates viral RNA dimerization.J. Mol. Biol. 2007; 371: 1084-1098Crossref PubMed Scopus (47) Google Scholar, 40.Ohishi M. Nakano T. Sakuragi S. Shioda T. Sano K. Sakuragi J. The relationship between HIV-1 genome RNA dimerization, virion maturation and infectivity.Nucleic Acids Res. 2011; 39: 3404-3417Crossref PubMed Scopus (29) Google Scholar, 43.Jalalirad M. Laughrea M. Formation of immature and mature genomic RNA dimers in wild-type and protease-inactive HIV-1: differential roles of the Gag polyprotein, nucleocapsid proteins NCp15, NCp9, NCp7, and the dimerization initiation site.Virology. 2010; 407: 225-236Crossref PubMed Scopus (28) Google Scholar). In general, not much is known about the actual RNA-RNA contacts during virion assembly and subsequent maturation. Possibly, this dimer maturation process is analogous to the stabilizing RNA rearrangements that are observed in vitro when the KL dimer is converted into the ED. Unfortunately, classical RNA probing studies cannot discriminate between the KL and ED conformations because the same nucleotides are involved in these intra- and intermolecular base pairing schemes. Therefore, the molecular nature of the structural transition from fragile to stable RNA dimers inside virions remains elusive. Moore et al. (24.Moore M.D. Fu W. Nikolaitchik O. Chen J. Ptak R.G. Hu W.S. Dimer initiation signal of human immunodeficiency virus type 1: its role in partner selection during RNA copackaging and its effects on recombination.J. Virol. 2007; 81: 4002-4011Crossref PubMed Scopus (80) Google Scholar) created DIS mutants that, at least in vitro, can form KL dimers, but not ED. Their experiments indicated that ED formation is not required for the initial RNA partner selection, subsequent RNA packaging, and the process of recombination. This does not exclude other roles for the ED in HIV-1 replication. The initial interaction between Gag and viral RNA during assembly and packaging has been studied in detail (44.Jouvenet N. Simon S.M. Bieniasz P.D. Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19114-19119Crossref PubMed Scopus (205) Google Scholar, 45.Kutluay S.B. Bieniasz P.D. Analysis of the initiating events in HIV-1 particle assembly and genome packaging.PLoS Pathog. 2010; 6: e1001200Crossref PubMed Scopus (146) Google Scholar). To investigate the role of ED formation on virus replication, we analyzed a set of HIV-1 variants with mutations that affect the DIS hairpin (13.Abbink T.E. Ooms M. Haasnoot P.C. Berkhout B. The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation.Biochemistry. 2005; 44: 9058-9066Crossref PubMed Scopus (92) Google Scholar). Virus replication was severely affected by alteration of a three-nucleotide motif (GGA triplet) immediately upstream of the DIS hairpin. Further analysis demonstrated that this mutation reduced in vitro ED formation and the amount of RNA dimers in virions. The crippled HIV-1 mutants were allowed to evolve by selection of faster replicating revertant viruses. Characterization of these variants confirmed the importance of the GGA triplet in both ED formation and viral replication. The 5′ leader mutations (J8, J9, and J10) were introduced in the HIV-1 molecular clone pLAI (46.Peden K. Emerman M. Montagnier L. Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI.Virology. 1991; 185: 661-672Crossref PubMed Scopus (318) Google Scholar) and the pLAI-R37 derivative (13.Abbink T.E. Ooms M. Haasnoot P.C. Berkhout B. The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation.Biochemistry. 2005; 44: 9058-9066Crossref PubMed Scopus (92) Google Scholar). pLAI-R37 has a deletion in the R-U5 region of the 3′ LTR. The WT 3′ LTR is restored during the reverse transcription process in the first round of virus replication (47.Berkhout B. van Wamel J. Klaver B. Requirements for DNA strand transfer during reverse transcription in mutant HIV-1 virions.J. Mol. Biol. 1995; 252: 59-69Crossref PubMed Scopus (51) Google Scholar). C33A human cervix carcinoma cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids (Invitrogen), 20 mm glucose, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2. The cells were grown in 2 cm2 wells and transfected with 1 μg of WT or mutant pLAI-R37 DNA by the calcium phosphate method, as previously described (48.Das A.T. Zhou X. Vink M. Klaver B. Verhoef K. Marzio G. Berkhout B. Viral evolution as a tool to improve the tetracycline-regulated gene expression system.J. Biol. Chem. 2004; 279: 18776-18782Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Two days post-transfection, the supernatant was harvested, and the cells were lysed in PBS containing 1% Empigen-BB. The intracellular and supernatant CA-p24 level was determined by ELISA (49.Jeeninga R.E. Jan B. van den Berg H. Berkhout B. Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias.Retrovirology. 2006; 3: 64Crossref PubMed Scopus (26) Google Scholar). SupT1 T cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2. Cells (10 × 106) were transfected with 250 ng or 1 μg of pLAI-R37 DNA by electroporation (250 V, 975 microfarad) using a Bio-Rad Gene Pulser II as previously described (48.Das A.T. Zhou X. Vink M. Klaver B. Verhoef K. Marzio G. Berkhout B. Viral evolution as a tool to improve the tetracycline-regulated gene expression system.J. Biol. Chem. 2004; 279: 18776-18782Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Cells were split 1 to 10 twice a week. The CA-p24 level in the culture medium was determined by ELISA. For virus evolution, 15 × 106 cells were transfected with 40 μg of pLAI-R37 DNA by electroporation and immediately split into six cultures. The protocol for virus evolution by prolonged cell-free passage of virus onto fresh, uninfected SupT1 cells was described previously (50.Berkhout B. Das A.T. Virus evolution as a tool to study HIV-1 biology.Methods Mol. Biol. 2009; 485: 436-451Crossref PubMed Scopus (11) Google Scholar). Isolation of total cellular DNA was performed by Tween 20/proteinase K treatment (51.Das A.T. Klaver B. Klasens B.I. van Wamel J.L. Berkhout B. A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication.J. Virol. 1997; 71: 2346-2356Crossref PubMed Google Scholar). The LTR leader region was PCR-amplified with primers T7-1 and TA014 (13.Abbink T.E. Ooms M. Haasnoot P.C. Berkhout B. The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation.Biochemistry. 2005; 44: 9058-9066Crossref PubMed Scopus (92) Google Scholar). The PCR products were sequenced directly, thus providing the population sequence of the viral quasispecies. PCR products were also cloned in the pCRII-TOPO vector (Invitrogen). The HindIII-ClaI fragment of selected clones was used to replace the corresponding fragment in pBlue-5′ LTR (52.Klaver B. Berkhout B. Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus.EMBO J. 1994; 13: 2650-2659Crossref PubMed Scopus (89) Google Scholar). The plasmid contains the XbaI-ClaI fragment of the infectious pLAI clone, including the 5′ LTR promoter sequence, the full-length leader sequence, and part of the Gag open reading frame (−454/+376, relative to the transcriptional start site at +1). Subsequently, the progeny XbaI-ClaI fragments were cloned into pLAI-R37. The constructs were verified by BigDye terminator sequencing (Applied Biosystems, Foster City, CA). TZM-bl cells (53.Wei X. Decker J.M. Liu H. Zhang Z. Arani R.B. Kilby J.M. Saag M.S. Wu X. Shaw G.M. Kappes J.C. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.Antimicrob. Agents Chemother. 2002; 46: 1896-1905Crossref PubMed Scopus (1340) Google Scholar, 54.Wei X. Decker J.M. Wang S. Hui H. Kappes J.C. Wu X. Salazar-Gonzalez J.F. Salazar M.G. Kilby J.M. Saag M.S. Komarova N.L. Nowak M.A. Hahn B.H. Kwong P.D. Shaw G.M. Antibody neutralization and escape by HIV-1.Nature. 2003; 422: 307-312Crossref PubMed Scopus (1991) Google Scholar) were maintained in DMEM supplemented with 10% FBS, nonessential amino acids (Invitrogen), 20 mm glucose, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2. TZM-bl cells were grown to 60% confluency in 2-cm2 wells and infected with WT or mutant C33A-produced virus (corresponding to 10 ng of CA-p24) in the presence of 80 μg/ml DEAE dextran. The cells were washed with PBS 2 days after infection and lysed in passive lysis buffer (Promega). The firefly luciferase was measured in cell lysates with the luciferase assay kit (Promega). The pLAI-based plasmids were used as template in a PCR with primers T7–2 and R:A368-A347 (complementary to nt +368 to +347 of the HIV-1 LAI genome with the transcription start site at +1). The PCR products were ethanol-precipitated and in vitro transcribed with the megashortscript T7 transcription kit (Ambion) in the presence of 1 μl of [α-32P]UTP (0.33 MBq/μl; PerkinElmer Life Sciences). Transcription reaction mixtures were incubated for 3 h at 37 °C, and the reactions were stopped by DNase treatment. The RNA was ethanol-precipitated, dissolved in water, and quantified by scintillation counting. To test the quality of the RNA, 20 ng of 32P-labeled RNA was incubated for 5 min at 85 °C in formamide-containing loading buffer (Ambion) and analyzed on a denaturing 6% polyacrylamide gel. Dimerization was performed with 20 ng of 32P-labeled RNA in 24 μl of dimerization buffer (83 mm Tris-HCl, pH 7.5, 125 mm KCl, and 5 mm MgCl2). The mixture was heated for 2 min at 85 °C, incubated for 10 min at 65 °C, and slowly cooled to room temperature for renaturing and dimerization. Samples were mixed with 12 μl of nondenaturing sample buffer (30% glycerol with bromphenol blue) and analyzed on nondenaturing 4% polyacrylam" @default.
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• W2038245782 date "2014-12-01" @default.
• W2038245782 modified "2023-09-30" @default.
• W2038245782 title "A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication" @default.
• W2038245782 cites W1480821243 @default.
• W2038245782 cites W1490329041 @default.
• W2038245782 cites W1500182662 @default.
• W2038245782 cites W1504782024 @default.
• W2038245782 cites W1507598539 @default.
• W2038245782 cites W1547414471 @default.
• W2038245782 cites W1653354461 @default.
• W2038245782 cites W1673789545 @default.
• W2038245782 cites W1936863780 @default.
• W2038245782 cites W1966320426 @default.
• W2038245782 cites W1970294872 @default.
• W2038245782 cites W1971135765 @default.
• W2038245782 cites W1973542661 @default.
• W2038245782 cites W1974729150 @default.
• W2038245782 cites W1975158050 @default.
• W2038245782 cites W1980779739 @default.
• W2038245782 cites W1984642176 @default.
• W2038245782 cites W1984724155 @default.
• W2038245782 cites W1985665571 @default.
• W2038245782 cites W1986665516 @default.
• W2038245782 cites W1989767992 @default.
• W2038245782 cites W1997677784 @default.
• W2038245782 cites W1999049218 @default.
• W2038245782 cites W2001453986 @default.
• W2038245782 cites W2001939628 @default.
• W2038245782 cites W2003181803 @default.
• W2038245782 cites W2007427182 @default.
• W2038245782 cites W2020790990 @default.
• W2038245782 cites W2021837455 @default.
• W2038245782 cites W2027051533 @default.
• W2038245782 cites W2027139827 @default.
• W2038245782 cites W2032136448 @default.
• W2038245782 cites W2032142885 @default.
• W2038245782 cites W2032467322 @default.
• W2038245782 cites W2037979460 @default.
• W2038245782 cites W2042638980 @default.
• W2038245782 cites W2042891181 @default.
• W2038245782 cites W2044595289 @default.
• W2038245782 cites W2047977722 @default.
• W2038245782 cites W2048057039 @default.
• W2038245782 cites W2050122573 @default.
• W2038245782 cites W2053837695 @default.
• W2038245782 cites W2054053883 @default.
• W2038245782 cites W2062413947 @default.
• W2038245782 cites W2066662883 @default.
• W2038245782 cites W2067145794 @default.
• W2038245782 cites W2073004681 @default.
• W2038245782 cites W2074339141 @default.
• W2038245782 cites W2077246276 @default.
• W2038245782 cites W2078419878 @default.
• W2038245782 cites W2098571862 @default.
• W2038245782 cites W2099147980 @default.
• W2038245782 cites W2105632217 @default.
• W2038245782 cites W2107194656 @default.
• W2038245782 cites W2115719885 @default.
• W2038245782 cites W2116450753 @default.
• W2038245782 cites W2117839471 @default.
• W2038245782 cites W2117867057 @default.
• W2038245782 cites W2130851381 @default.
• W2038245782 cites W2132762716 @default.
• W2038245782 cites W2135493913 @default.
• W2038245782 cites W2137583763 @default.
• W2038245782 cites W2138740229 @default.
• W2038245782 cites W2138819288 @default.
• W2038245782 cites W2141623970 @default.
• W2038245782 cites W2143642296 @default.
• W2038245782 cites W2144979082 @default.
• W2038245782 cites W2146478787 @default.
• W2038245782 cites W2149356773 @default.
• W2038245782 cites W2152114427 @default.
• W2038245782 cites W2153159177 @default.
• W2038245782 cites W2153641115 @default.
• W2038245782 cites W2154962388 @default.
• W2038245782 cites W2158977548 @default.
• W2038245782 cites W2160741454 @default.
• W2038245782 cites W2167337626 @default.
• W2038245782 cites W2419207896 @default.
• W2038245782 cites W89943134 @default.
• W2038245782 doi "https://doi.org/10.1074/jbc.m114.621425" @default.
• W2038245782 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4271197" @default.
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