Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor.

Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.

Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase.

A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer-template (P/T) sequences that bound to the vaccinia virus polymerase catalytic subunit (E9) with enhanced affinity. A single selected P/T sequence (referred to as E9-R5-12) bound in physiological salt conditions with an apparent equilibrium dissociation constant (KD,app) of 93 ± 7 nM. The dissociation rate constant (koff) and binding half-life (t1/2) for E9-R5-12 were 0.083 ± 0.019 min-1 and 8.6 ± 2.0 min, respectively. The values indicated a several-fold greater binding ability compared to controls, which bound too weakly to be accurately measured under the conditions employed. Loop-back DNA constructs with 3'-recessed termini derived from E9-R5-12 also showed enhanced binding when the hybrid region was 21 nucleotides or more. Although the sequence of E9-R5-12 matched perfectly over a 12-base-pair segment in the coding region of the virus B20 protein, there was no clear indication that this sequence plays any role in vaccinia virus biology, or a clear reason why it promotes stronger binding to E9. In addition to E9, five other polymerases (HIV-1, Moloney murine leukemia virus, and avian myeloblastosis virus reverse transcriptases (RTs), and Taq and Klenow DNA polymerases) have demonstrated strong sequence binding preferences for P/Ts and, in those cases, there was biological or potential evolutionary relevance. For the HIV-1 RT, sequence preferences were used to aid crystallization and study viral inhibitors. The results suggest that several other DNA polymerases may have P/T sequence preferences that could potentially be exploited in various protocols.

Comparison of the thermal stabilities of the αß heterodimer and the α subunit of avian myeloblastosis virus reverse transcriptase.

Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63-kDa α subunit and a 95-kDa ß subunit. In this study, we explored the role of the interaction between the α and ß subunits on AMV RT stability. The recombinant AMV RT α subunit was expressed in insect cells and purified. It exhibited lower thermal stability than the native AMV RT αß heterodimer. Unlike the αß heterodimer, the α subunit was not stabilized by template-primer. These results suggest that interaction between the α and ß subunits is important for AMV RT stability.

Myb-induced chromatin remodeling at a dual enhancer/promoter element involves non-coding rna transcription and is disrupted by oncogenic mutations of v-myb.

The oncogene v-myb of avian myeloblastosis virus (AMV) encodes a transcription factor (v-Myb) that transforms myelomonocytic cells by deregulating the expression of specific target genes. v-myb has acquired its oncogenic potential by truncation as well as by a number of point mutations of its cellular progenitor c-myb. As a result of these changes, the target gene spectrum v-Myb differs from that of c-Myb. We recently showed that the chicken mim-1 gene, a c-Myb target gene that is not activated by v-Myb harbors a powerful cell type-specific and Myb-inducible enhancer in addition to a Myb-responsive promoter. We now show that Myb-dependent activation of the mim-1 gene is accompanied by extensive remodeling of the nucleosomal architecture at the enhancer. We found that the mim-1 enhancer region also harbors a promoter whose activity is stimulated by Myb and which directs the transcription of an apparently non-coding RNA. Furthermore, our data show that the oncogenic mutations of AMV have disrupted the ability of v-Myb to induce remodeling of chromatin structure at the mim-1 enhancer. Together, our results demonstrate for the first time directly that Myb induces alterations of the nucleosomal organization at a relevant target site and provide new insight into the functional consequences of the oncogenic amino acid substitutions.

Analysis of the genetic complexity and abundance classes of messenger RNA in human liver and leukemic cells.

In an effort to determine the number of genes expressed as messenger RNA in disparate human tissues we have analyzed the genetic complexity of the polyribosome-associated poly(A)-containing RNA population obtained from liver and lymphoblastic leukemic cells. This was accomplished by measuring the kinetics of hybridization of mRNA to a complementary DNA probe synthesized by avian myeloblastosis virus reverse transcriptase in vitro. The results obtained from such an analysis revealed the presence of two major abundance classes of mRNA with a total genetic complexity of approximately 10,000 diverse mRNA species in both of these cell types. Diversity of mRNA species in these unrelated human cells was studied by heterologous hybridization reactions between the cDNA of one cell type and a vast excess of poly(A)-containing mRNA from another. These types of studies indicated that extensive homology (more than 80%) exists in the mRNA sequences of disparate human cell types and suggest that the vast majority of genetic information expressed as mRNA is required for the maintenance of cellular functions common to functionally different human tissues.

Effect of actinomycin D on nucleic acid hybridization: the cause of erroneous DNA elongation during DNA synthesis of RNA tumor viruses in vitro.

Actinomycin D, known for its suppression of cellular RNA synthesis and for the reduction of the rate of synthesis of double-stranded DNA by the RNA tumor virus RNA-dependent DNA polymerase, was found to interact with single-stranded DNA in such a way as to inhibit DNA . DNA and DNA . RNA hybridizations. This finding is discussed in the light of the observation that DNA elongation during DNA synthesis of RNA tumor viruses is blocked in vitro in the presence of actinomycin D. It thus supports the model that hybridization is a necessary step during RNA tumor virus DNA synthesis.

Synthesis of viral proteins by the avian myeloblastosis viral core component.

Avian Myeloblastosis Viral (AMV) core component was isolated and shown to synthesize AMV proteins in vitro. This reaction was linearly dependent on viral core concentration, proceeded linearly with time, and was inhibited by puromycin and aurintricarboxylic acid. The proteins synthesized in vitro co-electrophoresed and co-chromatographed with known proteins, and were immunoprecipitated by total and monospecific antibodies to known AMV proteins.

A cell-free mammalian protein-synthesizing system stimulated by RNA from avian myeloblastosis virus.

Carcinoma cells, oncornavirus-infected cells and fetal bovine tissue provide salt wash ribosomal factors capable of responding to avian myeloblastosis virus (AM virus)-RNA and stimulating the incorporation of amino acids into proteins as well as catalyzing the binding of N-acetylated (35S) methionyl-tRNA. The exogenously dependent amino acid incorporation system is stimulated by the high molecular weight species of AM virus-RNA only, particularly the fraction containing polyadenylate (poly(A)) residues; the system is also markedly inhibited by the low molecular weight AM virus-RNA species. Activity for the exogenous system displays very definite divalent/monovalent cation optima and requires the presence of mammalian transfer RNA.

Stepwise transition of aggregate structure of high-molecular-weight avian myeloblastosis virus RNA. Mode of releasing of associated 4S RNA.

Mode of releasing of associated 4S RNA species was studied during a controlled transition of aggregate structure of high-molecular-weight AMV-RNA. It has been found that associated 4S RNA constitutes 2.5% of 60S AMV-RNA complex. Approximately 60% of associated 4S RNA is successively released during treatment of viral RNA with increasing formamide concentration, concomitantly with the transition of 60S RNA aggregate through 50--55S RNA intermediate into the final 30--40S RNA subunits. 40% of 4S RNA remains associated with 30--40S RNA subunits prepared by formamide treatment and can be released from them by heating. A procedure is thus provided both for the isolation of oncornaviral RNA subunit structures deprived of various partions of associated 4S RNA and for the fractionation of 4S RNA species according to their binding affinity to the genome oncornaviral RNA.

In vitro processing of avian oncovirus precursor polypeptide Pr76gag by virion protein p15.

In vitro cleavage by p15 virion protein of the primary translation product of the avian oncovirus gag gene, the precursor polypeptide Pr76gag, was studied in kinetic experiments. Nondenatured 35S-methionine-labelled Pr76gag, which was synthetized in reticulocyte lysate programmed by genomic AMV-RNA was used as a substrate, pure native p15 (AMV) as a protease. Reaction conditions were optimal for in vitro protein synthesis. Composition of the cleavage products was estimated by immune precipitation with monospecific antisera against internal structural virion (AMV) proteins p27, p19, p15, and p12, and their size by SDS-PAGE. Monospecificity of each of the antisera was assessed by adsorption with the three respective heterologous gag proteins, followed by immune precipitation of 35S-methionine-labelled proteins of the virion (AMV) lysate. The p15-mediated in vitro cleavage proceeds rapidly and specifically. In the early stages (10 min incubation with p15) the Pr76gag was absent, and an optimum amount of six cleavage intermediates with the following size, composition (shown in parentheses) and orientation (determined form the presence or absence of antigenic determinants of p19 or p15 proteins as N- or C-terminal moieties of the precursor) was found: N-terminal fragments of 66K (p19, p27, p12) and 60K (p19, p27); internal fragments of 37K (p27, p12); C-terminal fragments of 51K (p27, p12, p15), 32K (p12, p15) and 21K (p12, p15). These intermediates were converted into the following four final cleavage products, as the only polypeptides detected after prolonged (5 h) incubations: mature p27 and mature p15 proteins and 38K and 34K polypeptides containing only p19 antigenic determinants. Mature p12 an p19 proteins were not found among cleavage products. Autocatalytic cleavage of Pr76gag was not observed. The results hav allowed us to conclude that the arrangement of the gag proteins in the Pr76gag is N-p19-(p10?)-p27-p12-p15-C and that under in vitro conditions p15 recognizes three correct cleavage sites on native Pr76gag: one located at p12-p15 junction and two on both sides of the p27 moiety, as well as aberrant cleavage sites located inside p12 and possibly also p10 moieties.

Binding properties of avian retroviral proteins. II. Binding of protein ASLV NC(p12) to viral RNA and proviral DNA.

Binding of the major avian retroviral nucleocapsid protein ASLV NC(p12) to the MAV-1 (myeloblastosis-associated virus) proviral dsDNA and viral ssRNA was analysed using electron microscopy. Specificity of the binding was estimated by special computer programs. The NC(p12) protein bound to MAV-1 proviral dsDNA (clone pAT153--MAV-1), but specificity of this binding was not found by computer evaluation. NC(p12) also bound to nondenatured 70S viral RNA at a rate of 25 +/- 3 molecules per RNA molecule. When this RNA was denatured either before or after the complexing, it showed no binding affinity for the protein. This result implies that preserved secondary structure of the viral RNA was required for the binding.

Binding properties of avian retroviral proteins. I. Preparation and basic characterization of ASLV NC(p12) and MA(p19).

Using SP-Sephadex column chromatography we isolated from an avian retrovirus, AMV(MAV), nucleic acid-binding proteins ASLV NC(p12) and MA(p19). As shown by several criteria, namely SDS-PAGE, PR(p15) protease activity, and nucleic acid binding assay with the use of both ss and ds DNAs, our NC(p12) and MA(p19) isolates are virtually pure proteins mutually not cross-contaminated. Rabbit anti-NC(p12) and anti-MA(p19) sera which we prepared did not cross-react mutually. We conclude that both NC(p12) and MA(p19) and antibodies against them are adequately pure preparations for investigating their nucleic acid binding specificities towards AMV(MAV) genomic RNA and MAV-1 proviral DNA using electron microscopy supported by computer analysis of electron micrographs.

Translation of genomic avian myeloblastosis virus RNA in a cell-free protein synthesis system from rabbit reticulocytes.

Reaction conditions suitable for translation of genomic avian myeloblastosis virus RNA in micrococcal nuclease-pretreated reticulocyte lysates are described. The products of translation were characterized by immunoprecipitation and gel electrophoresis and compared with virus-specific products formed in host cells. Genomic viral RNA directed in a cell-free system the synthesis of precursors to viral structural proteins, namely Pr76gag and Pr180gag,pol.