Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hausmann, Y. Articles by Rümenapf, T. Search for Related Content PubMed PubMed Citation Articles by Hausmann, Y. Articles by Rümenapf, T.

 Previous Article  |  Next Article 

Journal of Virology, May 2004, p. 5507-5512, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5507-5512.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Classical Swine Fever Virus Glycoprotein E^rns Is an Endoribonuclease with an Unusual Base Specificity

Yvonne Hausmann, Gleyder Roman-Sosa, Heinz-Jürgen Thiel, and Till Rümenapf^*

Institut für Virologie, Justus-Liebig-Universität, D-35392 Giessen, Germany

Received 2 October 2003/ Accepted 22 January 2004

Top Abstract Text References

 
The glycoprotein E^rns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for E^rns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5' of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that E^rns is a highly active endoribonuclease (k_cat/K_m = 2 x 10⁶ to 10 x 10⁶ M^–1 s^–1) with a strong affinity to NpU containing single-stranded RNA substrates (K_m = 85 to 260 nM).

Top Abstract Text References

 
Pestiviruses are small enveloped viruses with a single positive-stranded RNA genome and belong to the Flaviviridae (40). Presently the Pestivirus genus contains five species (3), four of which are important pathogens in farm animals, namely, classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV-1 and BVDV-2), and border disease virus (BDV) (40). One of the most remarkable features of pestiviruses is the virus-encoded glycoprotein E^rns, which has ribonucleolytic activity. CSFV E^rns was shown to be an essential structural component of the virion (41), and the RNase activity has been shown to be a determinant of the virulence of CSFV and BVDV-2 (25, 26). In addition to being a virus-associated protein, E^rns is secreted from CSFV-infected cells as soluble protein (32). E^rns is a highly N-glycosylated dimeric protein of 227 amino acids, which was typed as a member of the RNase T2 family based on a narrow sequence homology in the active-site domains [cassette 1, XXXHGL(I)WP; cassette 2, F(L)XXH(YD)EXXK(TR)HGT(AW)C] (12, 14, 15, 33, 42). The RNase T2 family encompasses a variety of secreted endoribonucleases with a distribution in all kingdoms (14, 15). RNases of the T2 type are usually larger than 200 amino acids and have no absolute base specificity but a preferred cleavage site. Most fungal T2 RNases cleave at Ap/N bonds, which is in accordance with the finding that in most ribonucleases the base preference is due to interactions with the base located at the 5'-terminal end (B1 site) (36). RNase MC1, which was isolated from bitter gourd (Momordica charantia) and which only encompasses 191 amino acids, also belongs to the T2 RNase family. It can be distinguished from fungal T2 RNases as well as from smaller RNases in RNase A and RNase T1 families because its base specificity is based on interactions with uridine residues at the 3'-terminal site (B2 site) (16, 29, 38).

Thus far E^rns has been shown to degrade polyuridylic acid (42), rRNA (12), and viral genomic RNA (12, 42) in vitro. It has been shown that E^rns binds to a variety of cells (13), and a C-terminal transport peptide was identified which translocates the protein through the plasma membrane (20). In order to define potential substrates of E^rns we have determined the requirements at the cleavage site in heteropolymeric substrates and the kinetic parameters of the RNase.

Recombinant CSFV E^rns, which was expressed in insect cells and purified by immunoaffinity chromatography, was used for limited digestion of a ³²P-labeled RNA substrate (4, 34). As substrate the polylinker sequence of pBS SKII was transcribed with T7 RNA polymerase and was radiolabeled at the 5' end with [-³²P]ATP and T4 polynucleotide kinase. Digests using 20 ng of E^rns, 2 U of RNase T₁, or 2 U of RNase PhyM and 10⁵ cpm (5'-³²P) of RNA substrate were performed under denaturing conditions (7 M urea, 1 mM EDTA, and 20 mM sodium-citrate, pH 6.0) for 15 min at 55°C in order to avoid secondary structure formation of the RNA substrate. The RNase digestions were subjected to denaturing gel electrophoresis in an analytical polyacrylamide gel (8% polyacrylamide [PAA], 7 M urea) side by side with a radiolabeled sequencing reaction mixture of the analogous cDNA region (Fig. 1). Comparison of the patterns generated by E^rns and the reference endoribonucleases RNase T₁ (Gp/N) and PhyM (Up/N, Ap/N) as well as the sequencing reaction revealed that cleavage by E^rns occurred 5' of uridine residues. Cleavage products were identified at all Np/U dinucleotide sequences (ApU, CpU, GpU, and UpU). E^rns retains activity in the presence of 7 M urea even at elevated temperatures (55°C), thus the enzyme is remarkably stable. To further determine the requirements at the NpU cleavage site, experiments with synthetic RNA oligonucleotides (5'GCCGACUUC3') were undertaken. Unfortunately these molecules were not cleaved by E^rns, because they were either too small or were uncleavable for other reasons. Therefore, larger single-stranded RNA substrates were designed as runoff transcripts from cDNA. To avoid the formation of secondary structures the substrate molecules consisted mainly of homopolymeric A. For this purpose oligo(dT/dA) adaptors were cloned into the HindIII/SacI sites of pBS SKII to form a cDNA sequence, which was devoid of uridine residues (substrate U [Sub-U]: 5[prime]GAACAAAAGCCGG-A₄₉-GC3'). The four possible NpU dinucleotides were introduced by site-directed mutagenesis 14 nucleotides (nt) upstream of the 3' end into Sub-U cDNA. Due to variable binding of the mutagenesis primers within the poly(A) sequence, the DNA templates led to synthesis of transcripts with different sizes (Sub-U, 64 nucleotides; Sub-ApU, 57 nucleotides; Sub-CpU, 46 nucleotides; Sub-GpU, 57 nucleotides; Sub-UpU, 53 nucleotides). RNA substrates were generated by using T3 RNA polymerase and were labeled either at the 5' end with [-³²P]ATP by T4-polynucleotide kinase (Sub-U [Fig. 2B]; Sub-ApU, not shown) or during transcription with [-³²P]UTP (Sub-ApU [Fig. 2A and C]). Probably due to the addition of nucleotides at the 3' end (5, 17, 28), the labeled in vitro transcripts resulted in products of different sizes and were therefore purified by denaturing gel electrophoresis. Molecules of appropriate sizes were excised from the gel and eluted in a buffer containing 10 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate (pH 7.5). Despite this purification, a mixture of substrate molecules was obtained which usually differed from each other by a single nucleotide (Fig. 2). For each reaction, 5 x 10⁴ cpm of the purified substrate was subjected to E^rns cleavage in 40 mM Tris-acetate, 0.5 mM EDTA (pH 6.0) (Fig. 2A and B). These buffer conditions have previously been described as optimal for E^rns activity (42). The uridine-containing substrate Sub-ApU was cleaved by E^rns in a concentration-dependent manner (Fig. 2A). This also applied to the substrates Sub-CpU, Sub-GpU, and Sub-UpU (data not shown). In contrast, Sub-U was resistant to cleavage by E^rns (Fig. 2B) but was degraded by RNase A (data not shown). The same cleavage pattern was observed when degradation was studied with lysates from PK15 cells which were infected with CSFV at a multiplicity of infection of 1 (Fig. 2C). For this purpose cells were lysed in 1% Triton X-100 24 h postinfection, and the equivalent of 10⁴ cells was used for the degradation of Sub-ApU in 40 mM Tris-acetate, 0.5 mM EDTA (pH 6.0) for the indicated times (Fig. 2C). No degradation of Sub-ApU was observed after incubation with lysates from PK15 cells infected with a recombinant CSFV, which encoded an enzymatically inactive E^rns (CSFV-E^rnsH₇₉D), or noninfected PK15 cells (Fig. 2C). Independent of the type of ³²P-labeling, the cleavage of Sub-ApU resulted in a set of 4 to 5 predominant cleavage products. The size of the largest cleavage product correlated to the position of the NpU bond in the substrate, irrespective of the position of the ³²P-label.

View larger version (79K): [in this window] [in a new window]

FIG. 1. Erns cleaves 5' of uridine residues. A T7 transcript encompassing the polylinker sequence of pBS SKII was labeled with [-32P]ATP at the 5' end and was incubated under conditions that prevented the degradation to go on to completion (Erns [20 ng], RNase T1 [2 U], or Phy M [2 U] in a solution of 7 M urea, 1 mM EDTA, 20 mM sodium-citrate [pH 6.0] at 55°C for 15 min). The reactions were subjected to high-voltage polyacrylamide gel electrophoresis in a denaturing 8% PAA sequencing gel side by side with a [-32P]ATP-labeled dideoxy sequencing reaction of the respective region of the plasmid (lanes are termed according to the dideoxy nucleotides T, A, G, and C). RNase T1 cleaves Gp/N, and RNase Phy M cuts preferentially at Ap/N and Up/N bonds. Cleavage by Erns occurs 5' of uridine residues.

View larger version (22K): [in this window] [in a new window]

FIG. 2. Erns endoribonuclease requires uridine residues in position B2. (A) [32P]UTP labeled Sub-ApU (5 x 104 cpm) was incubated without (lane 1) or with 100 ng (lane 2), 50 ng (lane 3), or 25 ng (lane 4) of purified Erns in 40 mM Tris-acetate, 0.5 mM EDTA (pH 6.0) at 37°C for 20 min. The reactions were subjected to high-voltage polyacrylamide gel electrophoresis in a denaturing 8% PAA sequencing gel. Sub-ApU appears as a doublet of 57 and 58 nucleotides due to purification of the RNA transcript. The predominant cleavage results in 32P-labeled products (PI) that are at least 14 nucleotides shorter than Sub-ApU, which corresponds to the position of the uridine residue. Cleavage products are unstable and are further degraded to oligonucleotides (PII). (B) 32P-labeled Sub-U (5 x 104 cpm) was incubated without (lane 1) or with 100 ng (lane 2) or 50 ng (lane 3) of purified Erns as described above and was analyzed on the same gel. RNA without a uridine residue does not serve as substrate for Erns. (C) 32P-labeled Sub-ApU (5 x 104 cpm) was incubated for 5, 15, 30, and 60 min without (lane 1) or with Triton X-100 lysates (1%) of 104 PK15 cells that were not infected (panel 3) or that were infected with CSFV (panel 2) or recombinant CSFV-ErnsH79D (panel 4) at a multiplicity of infection of 1. nt, nucleotides.

Although the cleavage occurs 5' of uridine, the labeled phosphate remains attached to the N base of the 5' cleavage product. RNase T2 and other RNases transfer the phosphate from the 5' position to the 2' hydroxyl group of the 5' product via the formation of a 2',3' cyclophosphate intermediate (14, 15, 30). Thus, the labeled phosphate remains linked to the 3' position of the ribose in the B1 site of the enzyme.

Errors which occur during transcription with T3 or T7 RNA polymerase are a probable reason for the origin of the heterogeneity of the labeled primary cleavage product (PI) product (Fig. 2). Addition of nontemplate nucleotides frequently occurs at the 3' end as well as at the 5' end (11, 31). The degradation assays revealed that the labeled 5' cleavage products (PI) were unstable and further degraded to oligonucleotides (PII). This degradation occurred in the absence of NpU because PI does not contain any uridine residue. That in fact two distinct cleavage events are catalyzed by E^rns became apparent by performing the degradation assay under denaturing conditions. Degradation of Sub-ApU with different amounts of E^rns (100, 30, 10, 3, and 1 ng) in a solution of 7 M urea, 1 mM EDTA, 20 mM sodium-citrate (pH 6.0) leads to an accumulation of PI (Fig. 3) while the same amount of enzyme in 40 mM Tris-acetate, 0.5 mM EDTA (pH 6.0) produced mainly PII. For these experiments the degradation reaction was separated on denaturing 8% PAA gels in a Mini Protean II apparatus (Bio-Rad). Because the uridine-free substrate Sub-U is not attacked by E^rns, there apparently is a difference in the nature of the substrate molecules. Possibly, the initial NpU specific cleavage modifies the PI product (i.e., by generating a 3' phosphate) so that it is recognized as a substrate. As an alternative it is conceivable that E^rns remains attached to PI after completion of the first cleavage and continues to cleave PI at random positions. This model could explain the stabilizing effect of urea on PI, because it likely reduces the affinity of E^rns to the NpU-free substrate more efficiently than the affinity to the NpU site. Determination of the precise mechanism of the second cleavage is the subject of future analysis.

View larger version (116K): [in this window] [in a new window]

FIG. 3. Separation of primary and secondary cleavage by urea. 32P-labeled Sub-ApU (5 x 104 cpm) was incubated with or without (w/o) Erns (100, 30, 10, 3, or 1 ng) for 15 min in nondenaturing (40 mM Tris-acetate, 0.5 mM EDTA [pH 6.0]) (A) or denaturing buffer (7 M urea, 1 mM EDTA, 20 mM sodium-citrate [pH 6.0]) (B) at 37°C. The reactions were electrophoretically separated in small denaturing polyacryamide gels. Due to the mini-gel format, the cleavage fragments PI and PII form discrete bands. The overall activity of Erns is threefold lower in urea than in Tris-acetate buffer. While in panel A the substrate is rapidly degraded into PII, panel B shows that the presence of 7 M urea stabilizes PI. Also shown is steady-state analysis of the endoribonucleolytic activity of Erns.

For the functional characterization of an enzyme and its substrate it is important to know the kinetic properties involved. Thus far, kinetic data for E^rns are only available for an UpU dinucleotide as substrate (42). To analyze whether the ribonucleolytic activity of E^rns in a heteropolymeric substrate depends on the nucleotide in the 5' position of NpU, the cleavage kinetics for the radiolabeled Sub-ApU, Sub-CpU, Sub-GpU, and Sub-UpU were determined. An E^rns degradation assay was developed in which 20, 40, 60, 80, and 100 ng of the unlabeled RNA substrates containing trace amounts (0.2%) of the same ³²P-labeled molecule were incubated with 10 ng of purified recombinant E^rns at 37°C for 10 to 60 s in 40 mM Tris-acetate, 0.5 mM EDTA (pH 6.0). The degradation reactions were stopped by addition of an equal volume of formamide, heated to 95°C and separated by electrophoresis in 8% PAA gels containing 7 M urea in a Mini Protean II apparatus (Bio-Rad). Each analysis was repeated at least three times. The amount of radioactivity per band was analyzed in a Fuji BAS1000 PhosphorImager. As an example, the degradation of 40 ng of Sub-GpU is shown in Fig. 4A. Because the amount of radioactivity (in counts per minute per nanogram) was known, the turnover of RNA substrate could be measured at all time points (T₀ to T₆₀). The kinetic parameters K_m and V_max of Sub-ApU, Sub-CpU, Sub-GpU, and Sub-UpU were calculated from the Lineweaver-Burk plots: 1/v versus 1/[s] (22), by using Sigmablot (SPSS-Science) (shown for GpU in Fig. 4B).

View larger version (24K): [in this window] [in a new window]

FIG. 4. (A) Forty nanograms of Sub-GpU, including trace amounts of 32P-labeled Sub-CpU, was incubated with 10 ng of Erns for 10, 20, 30, or 60 s at 37°C and was analyzed by electrophoresis in a small denaturing 8% PAA gel. The radioactivities of substrate and cleavage products were determined by phosphorimaging, and the counts per minute at t = 0 were equated with the amount of RNA substrate which allowed calculation of the turnover of RNA substrate at t = 10, 20, 30, and 60 s. The same experimental procedure was applied for 20, 60, 80, and 100 ng of Sub-GpU and the other substrates: Sub-ApU, -GpU, and -UpU. (B) Lineweaver-Burk analysis of values determined for the degradation of different concentrations of Sub-GpU Erns.

Determinations of the K_m values indicate that E^rns has a very high affinity (K_m, 84 x 10^–9 to 259 x 10^–9 M) for the tested substrates in the order UpU>GpU>CpU>ApU (Table 1). For Sub-UpU the affinity of E^rns is 1,000-fold higher than that for the dinucleotide UpU (K_m, 872.5 x 10^–6 ± 125.9 x 10^–6 M) (42). In contrast, the turnover of the substrates was fastest for Sub-ApU (151.2 fmol/s), which is three times faster than that determined for Sub-CpU. For UpU the results are ambiguous, because the cleavage site (ApUpU) actually allows two cleavages to occur. The catalytic activity of E^rns indicated by the k_cat/K_m values is considerably high (2 x 10⁶ to 1 x 10⁷ M^–1 s^–1) and operates near the limits of diffusion (10^–8 to 10^–9 M^–1 s^–1) (8).

View this table: [in this window] [in a new window]

TABLE 1. Steady-state kinetic parameters for cleavage of Sub-ApU, Sub-CpU, Sub-GpU, and Sub-UpU by Erns

E^rns is a highly active RNase of the T2 RNase family with an unusual cleavage site preference. Interestingly, the same cleavage preference (Np/U) was recently determined for RNase MC1, although there is very little sequence homology outside the active center. RNase MC1 cleaves phosphodiester bonds of A, C, G, or UpU, the most favorable substrate being CpU (16). Kinetic parameters of RNase MC1, which have been determined for CpU (K_m, 1.38 x 10³ ± 0.2 x 10³ M; k_cat, 746 ± 95 min^–1), and the k_cat/K_m value (542 x 10^–3 ± 50 x 10^–3 M^–1 min^–1) (29), reveal that E^rns has a 10⁷-fold higher activity at least with respect to the oligomeric substrates used in our analysis.

The aim of this study was to determine the substrate specificity of the RNase E^rns of CSFV. In particular, the question was addressed whether E^rns prefers distinct sequence motifs or substrates (i.e., 28S rRNA) as it is described for several secreted ribonucleases (i.e., onconase [21]; BS-RNase [18]; and -Sarcin, restrictocin, or mitogillin [24]) or targets a wide range of single-stranded RNA molecules. The kinetic data revealed that the substrate recognition of E^rns itself is not very discriminating, because the enzyme degraded the test substrates near its theoretical maximum. In other words, the affinity of E^rns for substrates containing the ubiquitous NpU cleavage is already so high that an even higher specificity for other (unknown) substrates is unlikely. Although the substrate specificity of E^rns was unaltererd by using PK15 cell lysates, it is possible that other factors define the actual targets of E^rns as it is reported for vhs RNase of alphaherpesviruses (35). vhs RNase, which is located in the tegument of the virion (19), is released during fusion at the plasma membrane and acts as an unspecific modulator of host cell translation by cleaving mRNAs (7, 37). Degradation of mRNAs by vhs RNase is initiated near the 5' end of target mRNAs (6) and requires a cellular factor (23).

It was recently shown that nonreplicative recombination in poliovirus requires 3'phosphate and 5'hydroxyl modifications of the joining RNA fragments (9, 10). Strikingly, these ends are produced by many ribonucleases, including RNases of the T2 family, and it is also evident that E^rns transfers the ³²P-labeled -phosphate of the uridine residue to the 3' end of the 5' fragment. While it is not shown whether nonreplicative recombination also applies for pestiviruses, recombinations of virus genomes with host cellular mRNAs (e.g., ubiquitin) occur with an unprecedented high frequency (1, 2, 27, 39). It is tempting to speculate that E^rns may even be advantageous for the virus survival and evolution by being mechanistically involved in the recombination of pestiviruses, but extensive future analyses are needed to test this hypothesis.

 
This work was funded by Deutsche Forschungsgemeinschaft, SFB 535. Y.H. was and G.R.-S. is a fellow of the Graduiertenkolleg "Biochemistry of Nucleoprotein-Complexes."

 
* Corresponding author. Mailing address: Institut für Virologie, Frankfurter Str. 107, D-35392 Giessen, Germany. Phone: 49-641-99-38356. Fax: 49-641-99-38359. E-mail: Till.H.Ruemenapf{at}vetmed.uni-giessen.de.

Top Abstract Text References

 

1
1. Baroth, M., M. Orlich, H. J. Thiel, and P. Becher. 2000. Insertion of cellular NEDD8 coding sequences in a pestivirus. Virology 278:456-466.[CrossRef][Medline]
2
2. Becher, P., G. Meyers, A. D. Shannon, and H. J. Thiel. 1996. Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome. J. Virol. 70:2992-2998.[Abstract]
3
3. Becher, P., R. A. Ramirez, M. Orlich, S. C. Rosales, M. König, M. Schweizer, H. Stadler, H. Schirrmeier, and H. J. Thiel. 2003. Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 311:96-104.[CrossRef][Medline]
4
4. Donis-Keller, H., A. M. Maxam, and W. Gilbert. 1977. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 4:2527-2538.[Abstract/Free Full Text]
5
5. Draper, D. E., S. A. White, and J. M. Kean. 1988. Preparation of specific ribosomal RNA fragments. Methods Enzymol. 164:221-237.[Medline]
6
6. Elgadi, M. M., C. E. Hayes, and J. R. Smiley. 1999. The herpes simplex virus vhs protein induces endoribocleolytic cleavage of target RNAs in cell extracts. J. Virol. 73:7153-7164.[Abstract/Free Full Text]
7
7. Everly, D. N., P. Feng, S. Mian, and G. S. Read. 2002. mRNA degradation by the virion host shutoff (vhs) protein of herpes simplex virus: genetic and biochemical evidence that vhs is a nuclease. J. Virol. 76:8560-8571.[Abstract/Free Full Text]
8
8. Fersht, A. 1999. Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding. W. H. Freeman & Co., New York, N.Y.
9
9. Gmyl, A. P., E. V. Belousov, S. V. Maslova, E. V. Khitrina, A. B. Chetverin, and V. I. Agol. 1999. Nonreplicative RNA recombination in poliovirus. J. Virol. 73:8958-8965.[Abstract/Free Full Text]
10
10. Gmyl, A. P., S. A. Korshenko, E. V. Belousov, E. V. Khitrina, and V. I. Agol. 2003. Nonreplicative homologous RNA recombination: promiscuous joining of RNA pieces? RNA 9:1221-1231.[Abstract/Free Full Text]
11
11. Helm, M., H. Brulé, R. Giegé, and C. Florentz. 1999. More mistakes by T7 RNA polymerase at the 5' ends of in vitro-transcribed RNAs. RNA 5:618-621.[CrossRef][Medline]
12
12. Hulst, M. M., G. Himes, E. Newbigin, and R. J. Moormann. 1994. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology 200:558-565.[CrossRef][Medline]
13
13. Hulst, M. M., and R. J. Moormann. 1997. Inhibition of pestivirus infection in cell culture by envelope proteins E(rns) and E2 of classical swine fever virus: E(rns) and E2 interact with different receptors. J. Gen. Virol. 78:2779-2787.[Abstract]
14
14. Irie, M. 1997. RNase T1/RNase T2 family RNases, p. 102-130. In G. D'Alessio and J. F. Riordan (ed.), Ribonucleases: structures and functions. Academic Press, New York, N.Y.
15
15. Irie, M., and K. Ohgi. 2001. Ribonuclease T2. Methods Enzymol. 341:42-56.[Medline]
16
16. Irie, M., H. Watanabe, K. Ohgi, Y. Minami, H. Yamada, and G. Funatsu. 1993. Base specificity of two plant ribonucleases from Momordica charantia and Luffa cylindrica. Biosci. Biotechnol. Biochem. 57:497-498.
17
17. Kholod, N., K. Vassilenko, M. Shlyapnikov, V. Ksenzenko, and L. Kisselev. 1998. Preparation of active tRNA gene transcripts devoid of 3'-extended products and dimers. Nucleic Acids Res. 26:2500-2501.[Abstract/Free Full Text]
18
18. Kim, J. S., J. Soucek, J. Matousek, and R. T. Raines. 1995. Mechanism of ribonuclease cytotoxicity. J. Biol. Chem. 270:31097-31102.[Abstract/Free Full Text]
19
19. Lam, Q., C. A. Smibert, K. E. Koop, C. Lavery, J. P. Capone, S. P. Weinheimer, and J. R. Smiley. 1996. Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function. EMBO J. 15:2575-2581.[Medline]
20
20. Langedijk, J. P. 2002. Translocation activity of C-terminal domain of pestivirus E(rns) and ribotoxin L3 loop. J. Biol. Chem. 277:5308-5314.[Abstract/Free Full Text]
21
21. Leland, P. A., and R. T. Raines. 2001. Review: cancer chemotherapy - ribonucleases to the rescue. Chem. Biol. 8:405-413.[CrossRef][Medline]
22
22. Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 76:658-666.
23
23. Lu, P., H. A. Saffran, and J. R. Smiley. 2001. The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity. J. Virol. 75:1072-1076.[Abstract/Free Full Text]
24
24. Makarov, A. A., and O. N. Ilinskaya. 2003. Minireview: cytotoxic ribonucleases: molecular weapons and their targets. FEBS Lett. 540:15-20.[CrossRef][Medline]
25
25. Meyer, C., M. von Freyburg, K. Elbers, and G. Meyers. 2002. Recovery of virulent and RNase-negative attenuated type 2 bovine viral diarrhea viruses from infectious cDNA clones. J. Virol. 76:8494-8503.[Abstract/Free Full Text]
26
26. Meyers, G., A. Saalmuller, and M. Buttner. 1999. Mutations abrogating the RNase activity in glycoprotein E(rns) of the pestivirus classical swine fever virus lead to virus attenuation. J. Virol. 73:10224-10235.[Abstract/Free Full Text]
27
27. Meyers, G., N. Tautz, P. Becher, H. J. Thiel, and B. M. Kümmerer. 1996. Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J. Virol. 70:8606-8613.[Abstract]
28
28. Milligan, J. F., D. R. Groebe, G. W. Witherell, and O. C. Uhlenbeck. 1987. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15:8783-8798.[Abstract/Free Full Text]
29
29. Numata, T., A. Suzuki, M. Yao, I. Tanaka, and M. Kimura. 2001. Amino acid residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity. Biochemistry 40:524-530.[CrossRef][Medline]
30
30. Ohgi, K., M. Iwama, K. Tada, R. Takizawa, and M. Irie. 1995. Role of Lys108 in the enzymatic activity of RNase Rh from Rhizopus niveus. J. Biochem. (Tokyo) 117:27-33.[Abstract/Free Full Text]
31
31. Pleiss, J. A., M. L. Derrick, and O. C. Uhlenbeck. 1998. T7 RNA polymerase produces 5' end heterogeneity during in vitro transcription from certain templates. RNA 4:1313-1317.[Abstract]
32
32. Rümenapf, T., G. Unger, J. H. Strauss, and H. J. Thiel. 1993. Processing of the envelope glycoproteins of pestiviruses. J. Virol. 67:3288-3294.[Abstract/Free Full Text]
33
33. Schneider, R., G. Unger, R. Stark, E. Schneider-Scherzer, and H. J. Thiel. 1993. Identification of a structural glycoprotein of an RNA virus as a ribonuclease. Science 261:1169-1171.[Abstract/Free Full Text]
34
34. Simoncsits, A., G. G. Brownlee, R. S. Brown, J. R. Rubin, and H. Guilley. 1977. New rapid gel sequencing method for RNA. Nature 269:833-836.[CrossRef][Medline]
35
35. Smiley, J. R., M. M. Elgadi, and H. A. Saffran. 2001. Herpes simplex virus vhs protein. Methods Enzymol. 342:440-451.[Medline]
36
36. Steyaert, J. 1997. A decade of protein engineering on ribonuclease T1-atomatic dissection of the enzyme-substrate interactions. Eur. J. Biochem. 247:1-11.[Medline]
37
37. Strom, T., and N. Frenkel. 1987. Effects of herpes simplex virus on mRNA stability. J. Virol. 61:2198-2207.[Abstract/Free Full Text]
38
38. Suzuki, A., M. Yao, I. Tanaka, T. Numata, S. Kikukawa, N. Yamasaki, and M. Kimura. 2000. Crystal structures of the ribonuclease MC1 from bitter gourd seeds, complexed with 2'-UMP or 3'-UMP, reveal structural basis for uridine specificity. Biochem. Biophys. Res. Commun. 275:572-576.[CrossRef][Medline]
39
39. Tautz, N., G. Meyers, and H. J. Thiel. 1993. Processing of poly-ubiquitin in the polyprotein of an RNA virus. Virology 197:74-85.[CrossRef][Medline]
40
40. Thiel, H. J., P. G. W. Plagemann, and V. Moennig. 1996. Pestiviruses, p. 1059-1073. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields Virology, 3rd ed. Lippincott-Raven Publishers, Philadelphia, Pa.
41
41. Widjojoatmodjo, M. N., H. G. van Gennip, A. Bouma, P. A. van Rijn, and R. J. Moormann. 2000. Classical swine fever virus E(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines. J. Virol. 74:2973-2980.[Abstract/Free Full Text]
42
42. Windisch, J. M., R. Schneider, R. Stark, E. Weiland, G. Meyers, and H. J. Thiel. 1996. RNase of classical swine fever virus: biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies. J. Virol. 70:352-358.[Abstract]

---

Journal of Virology, May 2004, p. 5507-5512, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5507-5512.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Tews, B. A., Schurmann, E.-M., Meyers, G. (2009). Mutation of Cysteine 171 of Pestivirus Erns RNase Prevents Homodimer Formation and Leads to Attenuation of Classical Swine Fever Virus. J. Virol. 83: 4823-4834 [Abstract] [Full Text]  
• Tews, B. A., Meyers, G. (2007). The Pestivirus Glycoprotein Erns Is Anchored in Plane in the Membrane via an Amphipathic Helix. J. Biol. Chem. 282: 32730-32741 [Abstract] [Full Text]  
• Meyers, G., Ege, A., Fetzer, C., von Freyburg, M., Elbers, K., Carr, V., Prentice, H., Charleston, B., Schurmann, E.-M. (2007). Bovine Viral Diarrhea Virus: Prevention of Persistent Fetal Infection by a Combination of Two Mutations Affecting Erns RNase and Npro Protease. J. Virol. 81: 3327-3338 [Abstract] [Full Text]  
• Fetzer, C., Tews, B. A., Meyers, G. (2005). The Carboxy-Terminal Sequence of the Pestivirus Glycoprotein Erns Represents an Unusual Type of Membrane Anchor. J. Virol. 79: 11901-11913 [Abstract] [Full Text]  
• Lin, M., Trottier, E., Pasick, J. (2005). Antibody Responses of Pigs to Defined Erns Fragments after Infection with Classical Swine Fever Virus. CVI 12: 180-186 [Abstract] [Full Text]  
• Ivanov, K. A., Hertzig, T., Rozanov, M., Bayer, S., Thiel, V., Gorbalenya, A. E., Ziebuhr, J. (2004). Major genetic marker of nidoviruses encodes a replicative endoribonuclease. Proc. Natl. Acad. Sci. USA 101: 12694-12699 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hausmann, Y. Articles by Rümenapf, T. Search for Related Content PubMed PubMed Citation Articles by Hausmann, Y. Articles by Rümenapf, T.