RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus

Alford, Raye Lynn; Honda, Sandra; Lawrence, Charles B.; Belmont, John W.

doi:10.1016/0042-6822(91)90990-s

Cited by 79 publications

(65 citation statements)

References 16 publications

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“…Chemical probing, computerized structure prediction, and mutational analyses all indicate that Mo-MuLV residues 215 to 420 can fold into at least three separate stem-loops (1,7,15,51). The most 3Ј of these, which we term DIS-2 (other authors refer to it as stem-loop B or H1), is centered around residues 283 to 298 and has been shown to be essential for dimerization in vitro (7,15,16) as well as to enhance the efficiency of genomic packaging into Mo-MuLV virions (12,34,35).…”

Section: Discussionmentioning

confidence: 99%

Bipartite Signal for Genomic RNA Dimerization in Moloney Murine Leukemia Virus

Parslow

2002

J Virol

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Retroviral virions each contain two identical genomic RNA strands that are stably but noncovalently joined in parallel near their 5 ends. For certain viruses, this dimerization has been shown to depend on a unique RNA stem-loop locus, called the dimer initiation site (DIS), that efficiently homodimerizes through a palindromic base sequence in its loop. Previous studies with Moloney murine leukemia virus (Mo-MuLV) identified two alternative DIS loci that can each independently support RNA dimerization in vitro but whose relative contributions are unknown. We now report that both of these loci contribute to the assembly of the Mo-MuLV dimer. Using targeted deletions, point mutagenesis, and antisense oligonucleotides, we found that each of the two stem-loops forms as predicted and contributes independently to dimerization in vitro through a mechanism involving autocomplementary interactions of its loop. Disruption of either DIS locus individually reduced both the yield and the thermal stability of the in vitro dimers, whereas disruption of both eliminated dimerization altogether. Similarly, the thermal stability of virion-derived dimers was impaired by deletion of both DIS elements, and point mutations in either element produced defects in viral replication that correlated with their effects on in vitro RNA dimerization. These findings support the view that in some retroviruses, dimer initiation and stability involve two or more closely linked DIS loci which together align the nascent dimer strands in parallel and in register.Retroviral virions each contain two copies of the singlestranded viral genomic RNA that are stably but noncovalently linked to form a homodimer (17,42). The dimeric genome is believed to promote retroviral replication and recombination by allowing reverse transcriptase to switch from one viral RNA strand to the other whenever it encounters a break in its template. Indirect evidence suggests that the dimerized chromosomes are bound together by direct RNA-RNA linkages along their entire lengths; this scenario could explain, for example, how such dimers remain intact even when both RNA strands are extensively nicked and all virion proteins have been removed (39). However, the nature and locations of the imputed dimer contacts are almost entirely unknown. Indeed, the only site of contact that has been characterized in detail is a short region, known as the dimer linkage site (DLS), that occurs near the 5Ј ends of many or all retroviral genomes (17, 42). The DLS was first defined by electron microscopic examination of dimers extracted from virions, and its appearance and location are well conserved among diverse retroviruses (2,22,28,37,47). It is typically the most stable site of linkage between strands (often the only such site that can survive denaturation in 75% formamide) and hence may be the first to form during dimer assembly in vivo. Viruses carrying mutations in the DLS have been found to contain anomalously dimerized or monomeric RNA (as judged by nondenaturing gel electrophoresis) and al...

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Section: Discussionmentioning

confidence: 99%

Bipartite Signal for Genomic RNA Dimerization in Moloney Murine Leukemia Virus

Parslow

2002

J Virol

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“…Numerous techniques, including computer prediction, chemical accessibility, and directed mutagenesis, have been used to determine the secondary structure of the MMLV ⌿ region (2,23,42) (Fig. 1C).…”

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confidence: 99%

RNA Sequences in the Moloney Murine Leukemia Virus Genome Bound by the Gag Precursor Protein in the Yeast Three-Hybrid System

Evans

Bacharach

Goff

2004

J Virol

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Encapsidation of the Moloney murine leukemia virus (MMLV) genome is mediated through a specific interaction between the major viral structural protein, Gag, and an RNA packaging signal, ⌿. Many studies have investigated this process in vivo, although the specific examination of the Gag-RNA interaction in this context is difficult due to the variety of other viral functions involved in virion assembly in vivo. The Saccharomyces cerevisiae three-hybrid assay was used to directly examine the interaction between MMLV Gag and ⌿. In this system, MMLV RNA regions exhibiting high-affinity Gag binding were mapped. All Gag-binding regions were located 3 to the viral splice donor sequence of the viral RNA transcript. No single short RNA sequence within ⌿ supported strong Gag interaction. Instead, an RNA comprised of nearly the entire ⌿ region was necessary to demonstrate an appreciable Gag interaction in the yeast three-hybrid system. These finding support the notion that two stem-loops (C and D) are not sufficient to form a core MMLV encapsidation signal.

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“…After initiation of these experiments, it was shown that site 243, despite being unpaired, is close to domain 1 and was shown to be protected from all chemical modification by the compounds dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (20). These results suggested that domain 1 pairs with another sequence that lies outside the sequence analyzed (20 In addition to analysis of the correlation between in vitro cutting and in vivo activities, different designs were used in this study: a multiple ribozyme (long antisense sequence with four ribozyme domains inserted at different sites), single ribozymes, and antisense sequence alone. From our in vitro cleavage data, the multiple ribozyme appeared to be more efficient in cleaving target substrate than the individual single ribozymes.…”

Section: Discussionmentioning

confidence: 99%

“…According to Alford et al (20), who described the RNA / packaging region of Mo-MLV, and our own data utilizing the Zuker folding program (14) (data not shown), sites 274 and 366 are within domains 4 and 6 of the RNA secondary structure of the Mo-MLV fpackaging region (20). These two domains are present in a segment of the packaging signal that appears to be absolutely required for qi function (21).…”

Section: Discussionmentioning

confidence: 99%

Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.

Sun

Warrilow

Wang

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Several hammerhead ribozymes targeted to different sites within the retroviral packaging () sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV 4epackaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV Or site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro deavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 packang site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a snficant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the * packaging site with hammerhead ribozymes.MLV is useful for assessing the efficacy of antiviral ribozymes because (i) it is an RNA virus, an ideal target for ribozyme action, (ii) levels can be assayed in tissue culture systems, and (iii) it induces two assessable phenotypes in mice-viremia and leukemia. Anti-Mo-MLV ribozymes can thus be tested for their ability to reduce virus in cells in culture and viremia and subsequent leukemia in animals. Among several important elements for retroviral, including HIV-1 and Mo-MLV, replication are cis-acting viral genomic sequences necessary for the specific packaging of viral RNA into virus particles (11,12). Recent studies have indicated that antisense RNA complementary to the Mo-MLV packaging sequence could inhibit virus replication-dependent leukemia induction in mice (13).In the present report, we have used Mo-MLV as a first step for assessing proof of principle for efficacy of anti-retroviral ribozymes and to show a correlation between in vitro cleavage of the Mo-MLV packaging sequence and in vivo suppression of Mo-MLV replication mediated by hammerhead ribozymes. In addition, we demonstrate that a ribozyme construct targeted to the HIV-1 qi packaging region can significantly inhibit HIV-1 infectivity and viral production in a target cell population. The present results show a feasible approach to the suppression of retroviral replication by targeting hammerhead ribozymes to the retroviral 4i packaging site.

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RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus

Cited by 79 publications

References 16 publications

Bipartite Signal for Genomic RNA Dimerization in Moloney Murine Leukemia Virus

Bipartite Signal for Genomic RNA Dimerization in Moloney Murine Leukemia Virus

RNA Sequences in the Moloney Murine Leukemia Virus Genome Bound by the Gag Precursor Protein in the Yeast Three-Hybrid System

Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.

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