effect of s-adenosyl homocysteine on in vitro rna synthesis by vesicular stomatitis virus (vsv) and...

54 EFFECT OF S-ADENOSYL HOMOCYSTEINE ON IN VITRO RNA SYNTHESIS BY VESICULAR

STOMATITIS VIRUS (VSV) AND A MUTANT OF VSV.

D.MARGARET HUNTland ROSHNl MEHTA 21 , Dept. of Microbiology and Immunology,

University of South Carolina, Columbia, SC 29208 and *Dept. of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216, U.S.A.

It has been reported previously that our stock of tsG16(1), a temperature- -

sensitive mutant of vesicular stomatitis virus (VSV), Indiana serotype,

overproduces polyadenylic acid (poly (A)) in vitro and that the RNA made by

this mutant contains much longer poly (A) tracts than RNA made in vitro by

wild-type (wt) VSV. Others have reported that mRNAs made in vitro in the -

presence of S-adenosyl homocysteine (SAH) have long poly (A) tracts. We

investigated whether polyadenylation by tsG16(1) was sensitive to the -

presence of SAH and

poly (A) tracts and

SAH was rather more

Glasgow) or another

found that SAH did cause an increase in the size of

that the increased polyadenylation in the presence of

than that observed with the parental wt (VSV Indiana- -

wt (VSV Indiana-San Juan), There also was a difference -

between the two wt strains in their response to SW. Taking advantage of this -

we were able to show by fractionation-reconstitution studies that the L

protein can modulate the response to the presence of SAH. The implications of

these results will be discussed. (Supported by NIH grant AI 18201.)

59 OBSERVATIONS OF THE RNA POLYMERASE OF VESICULAR STOMATITIS VIRUS : PH OPTIMUM, SULPHYDRYL REQUIREMENT AND ENHANCEMENT BY POLYAMINES

R. MASSEY, ROGER VANDEROEF and JOHN LENARD

UMDNJ-Rutgers Medical School, Piscataway, N.J. 08854, LJ.S.A

We have investigated several properties of the RNA polymerase

activity of isolated nucleocapsids of vesicular stomatitis virus. (1) The pH

optimum for the polymerase reaction has been reported variously as 8.3, 8.0

and 7.3, using only tris buffer (pka=8.3). We find a pH optimum of 7.8, using

three different buffers having widely different pka's. (2) Conflicting reports

have appeared concerning the sulphydryl requirement for polymerase activity.

We have observed partial or no sulphydryl requirement for polymerase activity

in detergent disrupted virions. Purified nucleocapsids have a strong sulphy-

dry1 requirement, however, and this becomes absolute after dialysis.

This suggests that the polymerase is a sulphydryl enzyme, and that sulphydryl

reagents in sufficient or nearly sufficient quantities are contained inside

intact virions. N-ethylmaleimide (1 x lO(-4)M., O") rapidly inactivates nucleo-

capsid polymerase with simple exponential kinetics, suggesting the involvement

of a single sulphydryl or class of sulphydryl groups in the enzyme action.

Nucleoside triphosphates protect to varying degrees from inactivation by

N-ethylmaleimide, suggesting that the required sulphydryl is at or near the

active site. (3) Polymerase activity of nucleocapsids and disrupted virions is

enhanced 2-4 fold by addition of spermine (0.1 mM) or spermidine (0.5 mM).

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