Identification of the 3C-protease-mediated 2A/2B and 2B/2C cleavage sites in the nonstructural polyprotein precursor of a dicistrovirus lacking the NPGP motif

Dicistroviruses have motifs for picornavirus 2C, 3C, and 3D proteins in their nonstructural polyprotein C-terminal region. The proteins from the nonstructural, N-terminal region of the polyprotein remain to be characterized. We have identified 3C-mediated cleavage sites in the N-terminal region of the nonstructural polyprotein of the dicistrovirus Plautia stali intestine virus (PSIV). The 2B/2C cleavage site mapped to amino acids (aa) 408–409 (QD). 2B/2C cleavage sites were suggested to be conserved in dicistroviruses. The most N-terminal PSIV cleavage site was aa 286–287 (QS). Including previous results, the polyprotein contains nine proteins arranged as follows: 2A, 2B, 2C, 3A, 3B1, 3B2, 3B3, 3C, and 3D.     

Yellow fever virus proteins NS2A, NS2B, and NS4B: identification and partial N-terminal amino acid sequence analysis

A series of fusion proteins corresponding to the hydrophobic ns2 and ns4 regions of yellow fever virus (YF) were generated in Escherichia coli using trpE fusion vectors. Antisera to ns2 and ns4 region fusion proteins recognize virus-specific proteins of 15 and 27 kDa, respectively. N-terminal amino acid sequence analysis of the 27-kDa protein indicates that the N-terminus of YF NS4B immediately follows a signalase-like cleavage site. Additional sequence data generated by microsequence analysis of labeled proteins immunoprecipitated with mouse hyperimmune antisera have identified the 15-kDa protein as NS2B and an additional 20-kDa viral protein as NS2A. Comparison of the sequences adjacent to the N-termini of these viral proteins suggests that three distinct types of cleavage events are involved in processing the hydrophobic YF ns2 and ns4 regions. These include cleavage after a short side chain amino acid to generate the N-terminus of NS2A, cleavage after two arginine residues to produce the N-terminus of NS2B, and a cleavage site consistent with the specificity of signalase to generate the N-terminus of NS4B. Analysis of virus-specific protein patterns in several different mammalian cell lines and in Aedes albopictus cells suggests that the same cleavage sites are used in different hosts. These findings are discussed in relation to the processing of flavivirus polyproteins.     

Substrate requirements of a human rhinoviral 2A proteinase.

The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2. This enzyme was shown to have an unusual preference at the P1 site; out of eight amino acid substitutions made, only the branched amino acids Val and Ile were not readily accepted. The HRV2 2A was shown to process poorly the HRV89 2A cleavage site and to be unable to cleave at sites which included the P' region of poliovirus or HRV14. Furthermore, the 2A of HRV89 preferred the cleavage site of HRV2 to its own.     

Amino acid sequence analysis of bovine rotavirus B223 reveals a unique outer capsid protein VP4 and confirms a third bovine VP4 type.

The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.     

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C^pro. Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.     

Characterization of the potyviral HC-pro autoproteolytic cleavage site.

The helper component-proteinase (HC-Pro) encoded by potyviruses functions to cleave the viral polyprotein by an autoproteolytic mechanism at the HC-Pro C-terminus. This protein belongs to a group of viral cysteine-type proteinases and has been shown previously to catalyze proteolysis between a Gly-Gly dipeptide. The amino acid sequence requirements surrounding the HC-Pro C-terminal cleavage site of the tobacco etch virus polyprotein have been investigated using site-directed mutagenesis and in vitro expression systems. A total of 51 polyprotein derivatives, each differing by the substitution of a single amino acid between the P5 and P2' positions, were tested for autoproteolytic activity. Substitutions of Tyr (P4), Val (P2), Gly (P1), and Gly (P1') were found to eliminate or nearly eliminate proteolysis. Substitutions of Thr (P5), Asn (P3), and Met (P2'), on the other hand, were permissive for proteolysis, although the apparent processing rates of some polyproteins containing these alterations were reduced. These results suggest that auto-recognition by HC-Pro involves the interaction of the enzymatic binding site with four amino acids surrounding the cleavage site. Comparison of the homologous sequences of five potyviral polyproteins revealed that the residues essential for processing are strictly conserved, whereas the nonessential residues are divergent. The relationship between HC-Pro and other viral and cellular cysteine-type proteinases is discussed.     

Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.

Poliovirus polypeptide 2C is a nonstructural protein involved in replication of the viral genome. Analysis of the primary amino acid sequence of 2C shows homology to a family of proteins which contain a nucleoside-triphosphate (NTP)-binding motif. This motif consists of elements "A" (2/5 hydrophobic stretch) G/AXXGXGKS/T, where X stands for any amino acid, and "B" (3/5 hydrophobic stretch) D or DD/E. To assess the significance of the consensus sequence in 2C, we have engineered point mutations into the most conserved residues in the A and B sites and tested their effect on viral RNA replication in vivo and translation in vitro. Whereas in vitro translation of synthetic RNAs carrying mutations in the NTP-binding motif showed efficient processing of all viral proteins, indistinguishable from that of the parental strain, transfection of the RNAs into HeLa cells did not give rise to infectious virus. No viral RNA replication could be detected in cells transfected with mutant RNAs. However, revertants to the wild-type genotype in the A and B sites were obtained which gave rise to wild-type RNA synthesis, but pseudorevertants or second-site suppressors were not observed. Thus, viral RNA synthesis is greatly reduced but not entirely abolished in cells transfected with mutant RNAs. These results strongly suggest a functional role for the proposed NTP-binding motif of 2C in RNA replication and proliferation of poliovirus.     

Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses

The genome sequence of tick-borne encephalitis (TBE) virus (Western subtype vaccine strain Neudoerfl) was determined. This extends the previously published sequence of the structural proteins to the nonstructural protein region and noncoding sequences at the 5'- and 3'-termini. The amino-termini of the individual proteins were assigned by comparison with other flavivirus sequences. Amino acid homology calculations between TBE virus and mosquito-borne flaviviruses were performed for all nonstructural proteins. An evolutionary tree based on protein NS1 is presented that reveals the molecular basis of relationships among flaviviruses. Tick-borne and mosquito-borne flaviviruses share a common hydrophilicity profile and also other features of their primary sequences, such as the presumably functional Gly-Asp-Asp sequence element within protein NS5. Other characteristics, such as the potential N-glycosylation sites of protein NS1 and a potential proteolytic cleavage site within protein NS4B, are conserved within the mosquito-borne group, but differ in the TBE virus sequence.     

Proteolytic processing of poliovirus polyprotein: elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis

The polypeptide 3CD of many poliovirus strains can be cleaved at two different amino acid pairs. The viral proteinase 3C and the viral polymerase 3D result from cleavage at a Gln-Gly pair by proteinase 3C, whereas cleavage at a Tyr-Gly pair by proteinase 2A yields the alternative products 3C' and 3D'. Specific mutations were introduced into the 3C'/3D' cleavage site in an infectious cDNA clone of poliovirus type 1 (Mahoney) by oligonucleotide-directed mutagenesis in order to investigate the role of 3C' and 3D' in viral proliferation and to obtain information about the cleavage specificity of 2Apro. Substitution of a threonine residue by an alanine residue at position -2 (P2) of this cleavage site abolished cleavage, whereas substitution of a tyrosine residue by a phenylalanine residue at amino acid position -1 (P1) of the cleavage site did not influence processing. Both mutated cDNA clones produced infectious viruses (T147A and Y148F) on transfection. The phenotypes of the mutant viruses were similar to that of the parental strain. We conclude that (i) 3C' and 3D' are not essential for virus replication, (ii) a Phe-Gly pair at the cleavage site can be cleaved by 2Apro, and (iii) a threonine residue in the P2 position of the cleavage site may be important in substrate recognition by 2Apro.     

The Hemagglutinin Cleavability of a Virulent Avian Influenza Virus by Subtilisin-like Endoproteases Is Influenced by the Amino Acid Immediately Downstream of the Cleavage Site

Many viral membrane glycoproteins are post-translationally processed by intracellular endoproteases such as subtilisin-like proteases. These proteases recognize a cleavage site sequence comprising basic amino acids positioned upstream of the cleavage site of the viral proteins. Here, we mutated the glycine residue immediately downstream of the cleavage site (P1) of hemagglutinin (HA) from a virulent avian influenza virus, A/turkey/Ontario/7732/66 (H5N9) (R-R-R-K-K-R/G), to examine the effect of this mutation on its clevability. Substitution of Gly with Ile, Leu, Val, or Pro, but not Ala, Asp, Phe, His, Ser, or Thr, resulted in substantial reduction of HA cleavage by endogenous endoproteases in CV-1 cells and by vaccinia-expressed PC6 and, albeit to a lesser extent, furin. We conclude that HA cleavage by subtilisin-like proteases is influenced by the downstream P1 amino acid in the absence of upstream cleavage site sequence alterations.     

Effect of low pH on the hepatitis A virus maturation cleavage

Cleavage of VP0 to VP2 via intramolecular scission is known as the viral maturation cleavage, as VP0 is found in immature particles, whilst VP2 is found in mature particles. The effect of low pH on the kinetics of hepatitis A virus (HAV) capsid protein VP0 cleavage in provirions was examined by Western blot analysis. VP0 scission was found to be dramatically enhanced under acidic conditions, similar to those encountered on entry of virus particles into the cell via endocytosis. The cleavage of VP0 to VP2 led to an increase in the specific infectivity of viral particles, indicating that mature virions are more infectious than immature provirions. The data are consistent with a model where conformational changes induced by low pH aid scission of VP0, and the increase in kinetics of VP0 cleavage may have relevance for viral uncoating, as only mature HAV particles are thought capable of uncoating within the host cell.     

Two proteinase activities in HCV polypeptide expressed in insect cells using baculovirus vector

Summary Processing of HCV viral precursor protein requires at least two viral proteinases, Cpro-1 and Cpro-2, in addition to cellular proteinases. The HCV polypeptide that covers the region for the two viral proteinase domains was expressed in insect cells using a baculovirus expression system. The two proteinase activities were demonstrated in the infected cells. The Cpro-1-dependent cleavage site was estimated from the amino acid sequence of the N-terminus of the processed product. Analyses of the susceptibilities of various mutants altered at position P 1 and P 1 of the putative cleavage site suggested that amino acid residues at these positions is not essential for recognition and cleavage by Cpro-1-dependent activity.     

Synthetic peptide substrates for the immunoglobulin A1 protease from Neisseria gonorrhoeae (type 2).

Neisseria gonorrhoeae secretes protease which inactive human immunoglobulin A1 (IgA1) by cleavage of specific peptide bonds in the hinge region. The type 2 IgA1 protease (EC 3.4.24.13) is secreted as a 169-kDa precursor which undergoes autoproteolysis at three sites (A, B, and C) to release the 106-kDa active form of the enzyme (J. Pohlner, R. Halter, K. Beyreuther, and T. F. Meyer. Nature [London] 325:458-462, 1987). Synthetic decapeptides consisting of five residues on each side of the three autoproteolytic cleavage sites and their potential pentapeptide catabolites were prepared by solid-phase synthesis. Cleavage of the decapeptides by the type 2 IgA1 protease from N. gonorrhoeae was monitored by high-performance liquid chromatography. Peptides homologous with the amino acid sequences around the B and C sites are cleaved by the IgA1 protease. Amino acid analysis and Edman degradation show that the cleavage products have both the composition and amino acid sequence which would be expected from cleavage at the predicted sites. Km values of 1.35 mM and 3.43 mM and kcat values of 280 pmol/h/U and 439 pmol/h/U for the site B and site C peptides, respectively, were determined. The catalytic efficiency (kcat/Km) for the synthetic substrates is about 10% of that reported for intact IgA1. Cleavage of the peptides is inhibited by IgA1 protease inhibitors such as the tetrapeptide substrate analog inhibitor HRP-48, human colostrum, and a peptide-boronate transition state inhibitor. An extract from an N. gonorrhoeae construct lacking active IgA1 protease failed to cleave the synthetic substrate, while an extract from the control construct which secretes active enzyme completely hydrolyzed the synthetic peptide. Neither the site A peptide nor synthetic decapeptides encompassing cleavage sites in the hinge region of IgA1 are hydrolyzed by IgA1 protease. These are the first synthetic substrates to be reported for any IgA1 protease.     

Intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures

In a susceptible cell, enteroviruses induce a vesiculated region (the "virus-induced vesicles") which is both the site of viral RNA synthesis as well as the site referred to morphologically, as the "cytopathic effect." Proteins of poliovirus (type I, Mahoney) were shown to migrate into the region of the virus-induced vesicles of infected HEp-2 cells. Five proteins (P2-5b, P3-4b, P3-6a, P3-7c, P3-9) were found to be associated with the vesicles themselves, either as intrinsic membrane protein (P3-9) or in a soluble form within the vesicles (P3-4b, P3-7c, and, partially, P3-6a) or bound to a DOC-resistant structure (P2-5b and a small amount of P3-6a). Partial inhibition of the cleavage of the viral polyprotein with ZnCl2 was used to alter the viral protein pattern within the cells. The data obtained indicate that P2-5b is the protein responsible for the formation of the virus-induced vesicles.