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HIV-1 RT/Dsrna Initiation Complex Prior to Nucleotide Incorporation

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HIV-1 RT/Dsrna Initiation Complex Prior to Nucleotide Incorporation Structure of HIV-1 RT/dsRNA initiation complex prior to nucleotide incorporation Kalyan Dasa,b,c,d,1,2, Sergio E. Martineza,b,c,d,1, Jeffrey J. DeStefanoe,f, and Eddy Arnolda,b,2 aCenter for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854; bDepartment of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854; cRega Institute, The Katholieke Universiteit Leuven, 3000 Leuven, Belgium; dDepartment of Microbiology, Immunology and Transplantation, The Katholieke Universiteit Leuven, 3000 Leuven, Belgium; eDepartment of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; and fMaryland Pathogen Research Institute, University of Maryland, College Park, MD 20742 Edited by John M. Coffin, Tufts University, Boston, MA, and approved February 19, 2019 (received for review August 16, 2018) The initiation phase of HIV reverse transcription has features that are Other tRNAs, when mutated to have sequence complementarity distinct from its elongation phase. The first structure of a reverse with PBS, are not efficient for HIV-1 reverse transcription initiation transcription initiation complex (RTIC) that trapped the complex after (9, 10). Posttranscriptional modifications of tRNALys3 have been incorporation of one ddCMP nucleotide was published recently reported to affect the formation of transcription initiation- [Larsen KP, et al. (2018) Nature 557:118–122]. Here we report a crystal competent vRNA–tRNALys3 complexes (11, 12). Nucleotide structure of a catalytically active HIV-1 RT/dsRNA complex that incorporation for initiation is almost 50-fold slower than that mimics the state of the RTIC before the first nucleotide incorporation. for reverse transcription elongation, and the processivity of RT The structure reveals that the dsRNA-bound conformation of RT is during elongation is four orders of magnitude higher in com- closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) parison with initiation (13). The published kinetic studies indicate and dsDNA; a hyperextended thumb conformation helps to accom- that slower nucleotide incorporation by the RTIC could be due to a modate the relatively wide dsRNA duplex. The RNA primer 3′ end is different structural state of the initiation complex compared with positioned 5 Å away from the polymerase site; however, unlike in an elongation complexes and the lower binding affinity of RT to dsRNA. NNRTI-bound state in which structural elements of RT restrict the The structural state might be responsible for a lower rate of catalytic movement of the primer, the primer terminus of dsRNA is not incorporation, and the lower binding affinity of dsRNA to RT would blocked from reaching the active site of RT. The observed structural reduce the stability of the RTICcomparedwithRTelongation changes and energetic cost of bringing the primer 3′ endtothe complexes. However, the stability and catalytic efficiency of an RTIC priming site are hypothesized to explain the slower nucleotide in- in vivo might be improved by the involvement of HIV nucleocapsid corporation rate of the RTIC. An unusual crystal lattice interaction of (NC) protein (14, 15), and other viral and cellular components. dsRNA with its symmetry mate is reminiscent of the RNA architecture Structures of HIV-1 RT have been studied extensively for the – Lys3 within the extended vRNA tRNA in the RTIC. This RT/dsRNA com- past 25 y since the first RT structures were reported (16, 17). plex captures the key structural characteristics and components of Structures of RT have revealed various functional and conforma- the RTIC, including the RT conformational changes and interactions tional states that have helped in (i) explaining multiple unique with the dsRNA primer-binding site region, and these features have functionalities of this enzyme, (ii) understanding the molecular implications for better understanding of RT initiation. mechanisms of drug resistance, and (iii) assisting drug design (18). reverse transcription initiation | RTIC | double-stranded RNA structure | Significance DNA polymerase | HIV reverse transcriptase RT is a key enzyme in the life cycle of HIV, and is targeted by IV-1 reverse transcriptase (RT) is responsible for copying multiple antiviral drugs. Although for most of its function RT Hthe retroviral single-stranded RNA (ssRNA) genome into – – ∼ binds a dsDNA or RNA DNA template primer substrate, initia- viral double-stranded DNA (dsDNA). While copying the 10-kb tion of reverse transcription involves binding of dsRNA. The HIV-1 ssRNA genome to make the viral dsDNA, RT uses RNA- and current study presents a structure of an RT/dsRNA complex that ∼ DNA-dependent DNA polymerization to incorporate 20,000 nu- has the basic components of a reverse transcription initiation cleotides. During most of the dsDNA synthesis, RT binds a dsDNA complex (RTIC). The unique structural features help understand – – or an RNA DNA template primer (1, 2). The mechanism of reverse the significantly slower rate of nucleotide incorporation by an transcription initiation, however, is different from that of the elon- RTIC compared with a catalytically efficient reverse transcription gation phase because initiation requires an external primer. Like elongation complex. This complex may help in designing new other retroviruses, HIV-1 recruits a specific host tRNA that anneals experiments for understanding the intricate and slow process of to the primer-binding site (PBS) of the viral genome (3). The PBS reverse transcription initiation. sequence is different for different members of the retrovirus family, and thereby, a specific retrovirus recruits a specific cognate tRNA. Author contributions: K.D. and E.A. designed research; K.D., S.E.M., J.J.D., and E.A. per- HIV recruits tRNALys3 to initiate DNA polymerization, and tRNA- formed research; S.E.M. and J.J.D. contributed new reagents/analytic tools; K.D. and E.A. analyzed data; and K.D. and E.A. wrote the paper. Lys3 is packaged into newly assembled viruses (4, 5). The 5′ untranslated region (UTR) of the HIV genome contains The authors declare no conflict of interest. the PBS sequence, which is 18 nucleotides long. The PBS sequence This article is a PNAS Direct Submission. is complementary to the 3′ end of tRNALys3, and both anneal to This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). form an 18-mer dsRNA. The annealed 18-mer dsRNA should bind Data deposition: The atomic coordinates and structure factors have been deposited in the the nucleic acid-binding cleft of RT to start minus-strand DNA Protein Data Bank, www.wwpdb.org (PDB ID code 6HAK). ′ Lys3 synthesis from the 3 end of the tRNA . Apart from 18-mer 1K.D. and S.E.M. contributed equally to this work. dsRNA, additional structural elements of tRNA and vRNA in- 2To whom correspondence may be addressed. Email: [email protected] or arnold@ teract to form a compact complex (6, 7). The overall structure of the cabm.rutgers.edu. – Lys3 vRNA tRNA complex and its interactions with RT beyond the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PBS–tRNA duplex appear to be critical for stabilizing the RT ini- 1073/pnas.1814170116/-/DCSupplemental. tiation complex (RTIC) (8) and for reverse transcription initiation. Published online March 22, 2019. 7308–7313 | PNAS | April 9, 2019 | vol. 116 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1814170116 Downloaded by guest on September 27, 2021 Structural study of the RTIC has been challenging, and the recent 3′ end is positioned away from both the priming (P) and N sites. outstanding single-particle cryo-EM structure by Puglisi and co- One important question is: How can the RTIC incorporate the first workers (19) provided the first glimpse of RTIC architecture. A nucleotide if the primer end is not positioned for catalysis? Here we modified tRNALys3 containing a cross-linkable N2-thioalkyl guanine report a crystal structure of an HIV-1 RT/dsRNA catalytic complex (G) base at the sixth position from the 3′ end (20) was annealed to at 3.95-Å resolution in which the 3′ end of the primer RNA strand the PBS region of a 101-nucleotide long 5′ UTR section of vRNA, is expected to be at the P site (Fig. 1B), a state competent for in- and the thioalkylguanine of the annealed tRNA–vRNA formed a corporation of the first nucleotide. disulfide cross-link with Q258C mutant RT, yielding a homogenous and stable RTIC complex for structural study. The cryo-EM study Results and Discussion generated an experimental density map [Electron Microscopy Data Structural Features of the RT/dsRNA Complex. We designed a 23- Bank (EMDB)-7032] for the RT/vRNA–tRNALys3 complex at 8-Å mer template/17-mer primer dsRNA to mimic the PBS region of resolution and a 4.5-Å resolution density map (EMDB-7031) for the duplex in the vRNA–tRNALys3 complex. The dsRNA has a the core RT/dsRNA (PBS region); a final model containing a cross-linkable thioalkyl-modified G base on the template oppo- dsRNA and an RT poly-Ala backbone was generated (PDB ID site to the sixth base from the primer 3′ end (Fig. 1B). Compared 6B19) by fitting the structural elements of RT to the 4.5-Å map. with the PBS–tRNALys3 duplex sequence, the C and G nucleo- The structures revealed the basic architecture and several important tides for the sixth base pair were switched between the template features of the RTIC; however, structural and functional studies of and primer for template cross-linking. The dsRNA was cross- different states of the RTIC are required for gaining broader in- linked to RT through a disulfide bond between the modified G sights into the mechanism of reverse transcription initiation. and the mutated thumb residue C258 in the Q258C mutant RT The cross-linking of dsRNA to RT was performed with primer (20, 21).
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