Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA

Abstract

A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNAbinding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo(dT)-cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.

Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity

“…Retroviral nucleocapsid proteins have been shown to bind to both DNA and RNA and appear to preferentially interact with ss sequences (Davis et al, 1976;Nissen-Meyer & Abraham, 1980). These proteins appear kinetically competent to destabilize or unwind a variety of doublestranded nucleic acids and can therefore be considered helix-destabilizing proteins Summers et al, 1992).…”

Immunological characterization of the low‐molecular‐weight dna binding protein of mouse mammary tumor virus

“…The DNA-binding proteins of MMTV, MPMV, R-MuLV, M7, SMRV, AKR and EIAV were purified by sequential procedures of gel filtration in guanidine-hydrochloride and affinity chromato-single-stranded D N A and membrane filtration graphy on single-stranded calf thymus D N A (Davis et al, 1976). MMTV gp 52 was purified by sepharose as previously described (Long et al, sequential chromatography on DEAE-cellulose and 1977).…”

Retrospective on the all-in-one retroviral nucleocapsid protein

“…The hallmark of all retroviral NC proteins, with the exception of Foamy viruses (Stenbak and Linial, 2004), is the presence of one or two small highly conserved CX 2 CX 4 HX 4 C (CCHC) motifs called zinc fingers (ZFs) or zinc knuckles that bind zinc ions with high affinity (Berg, 1986;Mely et al, 1991Mely et al, , 1996Summers et al, 1990Summers et al, , 1992, and that are flanked by unstructured basic sequences. In retrospect, some 38 years ago NC of alpha-and gammaretroviruses was first isolated from purified virions and considered to be an unspecific nucleic acid binding protein (NABP) with some preference for single stranded sequences within structured RNA domains (Davis et al, 1976).…”

“…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).…”