Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNA ^Trp Primer Annealing

Abstract

Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity

“…The previously computationally predicted structures of MΨ and PBS [6][7][8]55] are highly consistent with our experimentally determined secondary structure model of the RSV 5 -leader RNA. The structural features of stem loops SL-A, SL-B, SL-C and L3 are identical, although the nucleotides at the 3 -end of the previously predicted MΨ structure are part of a larger stem loop in our structure (Figure 1).…”

“…It is likely that tRNA primer annealing stabilizes the overall conformation of the PBS. Indeed, previous studies showed that the PBS undergoes significant structural changes and forms extensive intermolecular interactions upon primer annealing [55]. The tRNA Trp primer is found annealed to the PBS in protease-defective virions, indicating that the Gag precursor is capable of chaperoning the annealing [21].…”

“…The secondary structures of several structural motifs of RSV gRNA, such as MΨ and primer binding site (PBS), have been previously predicted computationally [6][7][8]55]. However, whether these structural features exist in the context of the full 5 -leader has not been probed experimentally.…”

Analysis of cis‐regulatory elements in the 5′ untranslated region of murine leukemia virus controlling protein expression

“…surrounding region forms a complex RNA secondary structure, which is important for viral replication (18)(19)(20)(21). The 5 leader sequence, which extends from the PBS to the AUG codon of gag, is complex, with specific sequences that have features of the secondary structure (22)(23)(24).…”

Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes

“…Ty1 and Ty3 exhibit a variation of this theme inasmuch as nucleotides defining the PBS are non-contiguous (8)(9)(10). However, despite these differences, a wealth of genetic and biochemical data indicates that long-range interactions between the tRNA primer and viral RNA genome contribute to the efficiency with which minus-strand DNA synthesis is initiated (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Alternate regions of complementarity between the tRNA primer and viral RNA genome include (i) nucleotides at the tRNA 5 0 terminus in feline immunodeficiency virus (20), (ii) TCC loop nucleotides in Rous sarcoma virus (12), (iii) D-loop nucleotides in Ty1 (14) and Ty3 (6), and (iv) TCC and anticodon loop nucleotides in HIV-1 and HIV-2 (11,13,15,16,18,19,(22)(23)(24)(25)(26)(27)(28)(29)(30).…”