Site Of 3C Research Articles

A genetic locus in mutant poliovirus genomes involved in overproduction of RNA polymerase and 3C proteinase

A mutagenic oligonucleotide cassette was used to introduce single and tandem amino acid substitutions into the proteinase 3C coding region of an infectious poliovirus type 1 cl)NA. The sites targeted for mutagenesis, residues 60, 61, and 66, are located within a putative helical loop structure which may be involved in substrate recognition by the enzyme. Fourteen viable 3C proteinase mutants were isolated. A Lys å Arg substitution at position 60 resulted in cold sensitivity for growth at 33°. Replacement of Lys 60 with lie, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 31) RNA polymerase than wild-type poliovirus. 3C-mediated processing of the remaining sites within the polyprotein was not noticeably affected. The overproduction of 3D is a consequence of more efficient processing of the carboxy-terminal Gin-Gly amino acid pair of 3C. Together with a previous report in which substitution of Val 54 with an Ala residue results in a poliovirus that produces decreased levels of 3D, these observations provide evidence that the putative loop region (residues 51–66) may be a functional domain involved in recognition of the carboxy-terminal Gln-Gly cleavage site of 3C.

Methods for the separation, purification and measurement of nine components of hemolytic complement in guinea-pig serum

C′1 precipitates from normal guinea-pig serum at ionic strength 0.04 and pH 7.5. It may be purified further by reprecipitation during dialysis against 0.005 m phosphate buffer, pH 7.5. C′3b precipitates at ionic strength 0.04-0.06 and pH 5.0. It is easily purified by elution from DEAE-cellulose at about 0.12 ionic strength. C′3c precipitates at ionic strength 0.02 and pH 5.5. It can be purified by elution from DEAE-cellulose at about 0.065 ionic strength. C′2 and C′3d remain soluble when (NH 4) 2SO 4 is added to about 2.2 m and then can be precipitated at about 3.0 m (NH 4) 2SO 4. These two components can be separated easily by chromatography on CM-cellulose. The sequence and the approximate ionic strength at which eight components elute from CM-cellulose is: C′2, 0.05; C′3f, 0.12; C′4, 0.13; C′3c, 0.135; C′3b, 0.14; C′3e, 0.145; C′3a, 0.18; and C′3d, 0.20. The sequence and ionic strength of elution from DEAE-cellulose is: C′3e, 0.03; C′3a, 0.035; C′3c and C′3f, 0.065; C′3d, 0.095; C′3b and C′4, 0.12. The yield of C′2 from DEAE-cellulose has been low. A substance in normal guinea-pig serum which inactivates the C′3c site on the intermediate complexes, EAb C′1,4,2,3c, EAb C′1,4,3c or EAb C′4,3c, elutes from DEAE-cellulose at about 0.05 to 0.06 ionic strength. Another substance which inactivates C′1 elutes from DEAE-cellulose at about 0.12 ionic strength.