The virion host shutoff protein (UL41) of herpes simplex virus 1 is an endoribonuclease with a substrate specificity similar to that of RNase A

Abstract

Earlier, our laboratory reported that purified glutathione S-transferase-virion host shutoff (GST-vhs) protein exhibited endoribonucleolytic activity in in vitro assays using as substrates in vitro-transcribed regions of IEX-1 mRNA. Here, we report that studies of the cleavage patterns of synthetic RNA oligonucleotides defined the activity of GST-vhs as being similar to that of RNase A. Thus, GST-vhs cleaved the RNA at the 3' end of single-stranded cytidine and uridine residues. Since the GST-mvhs nuclease-defective mutant protein failed to cleave the synthetic RNAs, the results unambiguously attribute the activity to vhs.

FIG. 1.

Comparison of the cleavage pattern of RNA 1 produced by GST-vhs fusion protein with those generated by RNases A, T1, and V1. (A) Secondary structure of the RNA 1 oligonucleotide as predicted by the mfold program. (B) Either GST (0.1 μg/μl; lanes 1 to 4) or GST-vhs fusion protein (0.1 μg/μl; lanes 5 to 8) as well as two different concentrations of RNase A (0.1 μg/μl, lanes 9 and 10; 0.01 μg/μl, lanes 11 and 12) and RNase V1 (0.1 U/μl, lanes 13 and 14; 0.01 U/μl, lanes 15 and 16) were incubated with 5′-end-labeled RNA 1 substrate. Aliquots were removed at the indicated times (in minutes; top of gel) and analyzed as described in the text. (C) Either GST (0.1 μg/μl; lanes 2 to 5) or GST-vhs fusion protein (0.1 μg/μl; lanes 6 to 9) as well as RNase A (0.005 μg/μl; lanes 10 and 11) and RNase T1 (0.5 U/μl; lanes 12 and 13) were incubated with 5′-end-labeled RNA 1 substrate for the time intervals (in minutes; top of gel) shown and analyzed as for panel B. Lane 1, 5′-end-labeled Decade RNA markers. The lengths of the fragments (in nucleotides) are reported on the side.

FIG. 2.

GST-vhs fusion protein, but not GST-mvhs mutant, cleaves RNAs 2 and 3 similarly to RNase A. (A) Secondary structure of either RNA 2 (left) or RNA 3 (right) oligonucleotides as predicted by the mfold program; the arrows mark the sites of RNase A expected cleavage. (B) GST-vhs fusion protein (lanes 3, 4, 12, and 13), GST alone (lanes 5, 6, 14, and 15), or GST-mvhs mutant protein bearing alanine substitutions for E192, D194, and D195 residues (lanes 7, 8, 16, and 17) (each at 0.1 μg/μl) as well as 0.01 μg/μl RNase A (lanes 9, 10, 18, and 19) were incubated with either 5′-end-labeled RNA 2 substrate (lanes 3 to 10) or 5′-end-labeled RNA 3 substrate (lanes 12 to 19) for the time intervals shown (in minutes) and analyzed as described in the text. Lane 1, 5′-end-labeled Decade RNA marker. The lengths of the fragments (in nucleotides) are reported on the side. RNA 2 (lane 2) and RNA 3 (lane 11) were incubated for 15 min (15′) at 95°C in the presence of Ambion alkaline hydrolysis buffer (50 mM sodium carbonate [pH 9.2], 1 mM EDTA) in the attempt to generate a ladder of hydrolyzed RNA fragments.

FIG. 3.

GST-vhs fusion protein cleaves RNAs 4, 5, and 6 similarly to RNase A. (A) Left, secondary structure of RNA 4 oligonucleotide as predicted by the mfold program; the arrow marks the site of RNase T1 expected cleavage. Right, 0.1 μg/μl of GST-vhs fusion protein (lanes 2 and 3), GST alone (lanes 4 and 5), or GST-mvhs mutant protein (lanes 6 and 7) as well as 0.01 μg/μl RNase A (lanes 8 and 9) and 0.01 U/μl of RNase (T1) were incubated with 5′-end-labeled RNA 4 for the time intervals shown (in minutes; top of gel) and analyzed as described in the text. Lane 1, 5′-end-labeled Decade RNA marker. The lengths of the fragments (in nucleotides) are reported on the side. (B) Left, secondary structure of RNA 5 oligonucleotide as predicted by the mfold program; the arrow marks the site of RNase A expected cleavage. Right, 0.1 μg/μl of GST-vhs fusion protein (lanes 2 and 3), GST alone (lanes 4 and 5), or GST-mvhs mutant protein (lanes 6 and 7) as well as 0.01 μg/μl RNase A (lanes 8 and 9) and 0.01 U/μl of RNase (T1) were incubated 5′-end-labeled RNA 5 for the time intervals shown (in minutes; top of gel) and analyzed as for panel A. Lane 1, 5′-end-labeled Decade RNA marker. The lengths of the fragments (in nucleotides) are reported on the side. (C) Left, secondary structure of RNA 6 oligonucleotide as predicted by the mfold program; the arrow marks the site of RNase A expected cleavage. Right, 0.1 μg/μl of GST-vhs fusion protein (lanes 1 and 2), GST alone (lanes 3 and 4), or GST-mvhs mutant protein (lanes 5 and 6) as well as 0.01 μg/μl RNase A (lanes 7 and 8) were incubated with 5′-end-labeled RNA 6 substrate for the time intervals shown (in minutes; top of gel), and the samples were analyzed as for panels A and B.

FIG. 4.

GST-vhs fusion protein cleaves RNAs 7 and 8 similarly to RNase A. (A and B) Left, secondary structure of either RNA 7 (A) or RNA 8 (B) oligonucleotides as predicted by the mfold program; the circle marks the sites of RNase A predicted cleavage. Right, 0.1 μg/μl of GST-vhs fusion protein (lanes 2 and 3), GST alone (lanes 4 and 5), or GST-mvhs mutant protein (lanes 6 and 7) as well as 0.01 μg/μl RNase A (lanes 8 and 9) were incubated with either 5′-end-labeled RNA 7 substrate (A) or 5′-end-labeled RNA 8 substrate (B) for the time intervals shown (in minutes). Lane 1, 5′-end-labeled Decade RNA marker. The lengths of the fragments (in nucleotides) are reported on the side.