cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA

Abstract

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and transacting factor, nucleocapsid protein, and thus might be linked during virion formation. Cells infected with Moloney murine leukemia virus (Mo-MuLV) produce three major size classes of virus-specific RNAs that are the 22S, 35S, and 70S RNAs (5). Most of the 22S and 35S RNA is found in free cytoplasm, while most of the 70S RNA is in the membrane-bound fraction (9). The MoMuLV 22S RNA is the messenger for the Pr85env precursor and is not found in virions. The 35S RNA is the genome-length RNA of 8,332 nucleotides (nt) which assumes two functions: it is the messenger RNA for the Pr659'9 and

Engineering RNA for Targeted siRNA Delivery and Medical Application

“…Non-templated assembly involves the formation of a larger structure by individual components without any influence from external forces. Included in the non-templated category are ligation, chemical conjugation, covalent linkages, loop/loop interactions of RNAs and the formation of macromolecules by structural interaction in the formation of multimers [12,54,[135][136][137][138]. The use of bacteriophage phi29 pRNA as a building block for the construction of micrometre-sized RNA arrays for potential tissue and wound repair has been reported [11].…”

Retrospective on the all-in-one retroviral nucleocapsid protein

“…The interactions between NC protein and the genomic RNA in alpha-(RSV) and gamma-RV (MoMuLV) virions were initially examined using UV light at 252 nm for cross-linking since this approach allows the specific spatial relationship of certain amino acids and RNA bases to be characterized (see Burd and Dreyfuss, 1994). Results showed that NC was by far the major protein tightly bound to the genomic 70S RNA in the nucleocapsid (Darlix and Spahr, 1985;Meric et al, 1984;Prats et al, 1990;Tanchou et al, 1995). But these results were obscured and a matter of intense debate because MAp19 protein of RSV and pp12 protein of MoMuLV were originally reported to be the viral proteins tightly linked to the genome in virions (Darlix and Spahr, 1982;Sen et al, 1977).…”

“…The NC cross-linked RNA fragments were recovered and upon sequencing it was found that NC tightly binds a small number of genomic RNA sites rich in U and G residues, located in the multifunctional 5 untranslated leader region (5 UTR) (Dannull et al, 1994;Darlix and Spahr, 1982;Meric et al, 1984;Tanchou et al, 1995). This was of special interest since these sites are close to the dimer linkage structure (DLS) (Darlix and Spahr, 1982;Prats et al, 1990) that holds together the two genomic RNA in the virion nucleocapsid (Murti et al, 1981), and the DLS is part of the packaging psi signal (Pugatsch and Stacey, 1983). These findings suggested that NC interaction with high affinity sites presents in the 5 leader region, close to the DLS, might be required for the selective packaging of the genomic RNA during virus morphogenesis (Aldovini and Young, 1990;Lever et al, 1989).…”

“…Originally NC proteins were purified from RV virions (Davis et al, 1976;Henderson et al, 1981Henderson et al, , 1988Henderson et al, , 1990Henderson et al, , 1992Hizi et al, 1987;Prats et al, 1988Prats et al, , 1990 and later from recombinant Escherichia coli or synthesized in vitro by chemical methods (see below) and found to bind a wide variety of nucleic acids (NA) molecules with a preference for the genomic 70S RNA (Darlix and Spahr, 1985;Wu et al, 1996). Accordingly, the relative affinity of in vitro synthesized NC protein for retroviral RNA, DNA and oligonucleotides is in the order 'retroviral RNA > ssDNA > dsDNA > oligonucleotides' where the apparent affinity constant for the genomic RNA is 5 × 10 7 M −1 , about 25 times higher than that for poly(rA) (You and McHenry, 1993).…”

The role of proteolytic processing in the morphogenesis of virus particles

“…PR-defective particles are stable in conditions that disrupt mature virions (Stewart et al118 and references therein) which suggests that one function of PR may be to facilitate disassembly of virus particles after uptake into a cell. The involvement of processing in RNA dimerization may be important in viral replication, since a role for the dimeric RNP complex in reverse transcription has been postulated (Prats et al96 and references therein). Moreover, it is apparent that replicative enzymes such as RT are activated by proteolytic removal of inhibitory flanking domains (Le Grice et al 73 and Stewart et al 118, and references therein).…”