N-terminal protease of pestiviruses: identification of putative catalytic residues by site-directed mutagenesis.

Pestiviruses are the only members of the Flaviviridae that encode a nonstructural protease at the N terminus of their polyproteins. This N-terminal protease (Npro) cleaves itself off of the nascent polyprotein autocatalytically and thereby generates the N terminus of the adjacent viral capsid protein C. In previous reports, sequence similarities between Npro and the catalytic residues of papain-like cysteine proteases were put forward. To test this hypothesis, substitutions of cysteine and histidine residues within Npro were carried out by site-directed mutagenesis. Translation of the mutagenized Npro-C proteins in cell-free lysates confirmed that only the predicted Cys69 was an essential amino acid for proteolysis, not His130. Further essential residues were identified with His49 and Glu22. While it remains speculative whether Glu22-His49-Cys69 actually build a catalytic triad, these results invalidate the assumption that Npro is a papain-like cysteine protease.

Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses.

Western equine encephalomyelitis (WEE) virus (Togaviridae: Alphavirus) was shown previously to have arisen by recombination between eastern equine encephalomyelitis (EEE)- and Sindbis-like viruses (C. S. Hahn, S. Lustig, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 85:5997-6001, 1988). We have now examined the recombinational history and evolution of all viruses belonging to the WEE antigenic complex, including the Buggy Creek, Fort Morgan, Highlands J, Sindbis, Babanki, Ockelbo, Kyzylagach, Whataroa, and Aura viruses, using nucleotide sequences derived from representative strains. Two regions of the genome were examined: sequences of 477 nucleotides from the C terminus of the E1 envelope glycoprotein gene which in WEE virus was derived from the Sindbis-like virus parent, and 517 nucleotide sequences at the C terminus of the nsP4 gene which in WEE virus was derived from the EEE-like virus parent. Trees based on the E1 region indicated that all members of the WEE virus complex comprise a monophyletic group. Most closely related to WEE viruses are other New World members of the complex: the Highlands J, Buggy Creek, and Fort Morgan viruses. More distantly related WEE complex viruses included the Old World Sindbis, Babanki, Ockelbo, Kyzylagach, and Whataroa viruses, as well as the New World Aura virus. Detailed analyses of 38 strains of WEE virus revealed at least 4 major lineages; two were represented by isolates from Argentina, one was from Brazil, and a fourth contained isolates from many locations in South and North America as well as Cuba. Trees based on the nsP4 gene indicated that all New World WEE complex viruses except Aura virus are recombinants derived from EEE- and Sindbis-like virus ancestors. In contrast, the Old World members of the WEE complex, as well as Aura virus, did not appear to have recombinant genomes. Using an evolutionary rate estimate (2.8 x 10(-4) substitutions per nucleotide per year) obtained from E1-3' sequences of WEE viruses, we estimated that the recombination event occurred in the New World 1,300 to 1,900 years ago. This suggests that the alphaviruses originated in the New World a few thousand years ago.

Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7.

The pestivirus genome encodes a single polyprotein which is subject to co- and posttranslational processing by cellular and viral proteases. The map positions of all virus-encoded proteins are known with the exception of a hypothetical peptide (p?) which interlinks the glycoprotein E2 and the nonstructural protein NS2-3 approximately between amino acid positions 1060 and 1130. Expression studies with recombinant vaccinia viruses bearing a set of C-terminally truncated E2-p?-NS2-encoding sequences derived from a bovine viral diarrhea virus (BVDV) strain led to the identification of a minor fraction of E2 which had an increased molecular mass due to a C-terminal extension. This larger form of E2 (E2p7) was specifically recognized by an antiserum raised against the amino acid sequence from 1065 to 1125. In addition, the antibodies revealed a BVDV-encoded 7-kDa protein (p7) in infected cells. By radiosequencing it was determined that Val-1067 was the N-terminal amino acid of in vitro-synthesized p7. Analyses of BVDV and classical swine fever virus virions suggest that neither p7 nor E2p7 is a major structural constituent.

Using ubiquitin to follow the metabolic fate of a protein.

We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.

Classical swine fever virus: recovery of infectious viruses from cDNA constructs and generation of recombinant cytopathogenic defective interfering particles.

The 5'- and 3'-terminal sequences of the genomic RNA from classical swine fever virus (CSFV) were determined, and the resulting information was used for construction of full-length CSFV cDNA clones. After transfection of in vitro-transcribed RNA derived from a cDNA construct, infectious CSFV was recovered from porcine cells. To confirm the de novo generation of infectious CSFV from cloned DNA, a genetically tagged CSFV was constructed. In comparison with parental CSFV, the recombinant viruses were retarded in growth by about 1 order of magnitude. Introduction of a deletion by exchange of part of the full-length construct for corresponding cDNA fragments derived from the genomes of cytopathogenic CSFV defective interfering particles (DIs) (G. Meyers and H.-J. Thiel, J. Virol. 69:3683-3689. 1995) resulted in recovery of cytopathogenic DIs in the DI genomes is responsible for their cytopathogenicity. The established system will allow novel approaches to analysis of pestiviral molecular biology and in particular to elucidation of the molecular basis of attenuation and cytopathogenicity of these viruses.

Aura virus is a New World representative of Sindbis-like viruses.

Aura virus is an alphavirus present in Brazil and Argentina that is serologically related to Sindbis virus (present throughout the Old World) and to Western equine encephalitis (WEE) virus (present in the Americas). We have previously shown that WEE is a recombinant virus whose glycoproteins and part of whose 3' nontranslated region (NTR) are derived from a Sindbis-like virus, but the remainder of whose genome is derived from Eastern equine encephalitis (EEE) virus. We show here that Aura virus is a Sindbis-like virus that shares considerable organizational and sequence identity with Sindbis virus. Certain nucleotide sequence elements present in Aura RNA that are believed to function as promoters are almost identical to their Sindbis counterparts, repeated elements in the 3' nontranslated region are shared with Sindbis virus, and important antigenic epitopes are conserved between the two viruses. Despite their close relationship, the two viruses have diverged significantly, sharing 73% amino acid sequence identity in the nonstructural proteins and 62% identity in the structural proteins. This is about the same as the identities between EEE and Venezuelan equine encephalitis virus, whose promoter elements, 3' NTRs, and antigenic epitopes have diverged more radically, such that these two viruses are considered to belong to different subgroups. Importantly, the glycoproteins of WEE are more closely related to those of Sindbis than to those of Aura virus. From this we propose that an ancestral Sindbis-like virus present in the Americas (probably South America) diverged 1000-2000 years ago into a lineage that gave rise to Aura virus and a lineage that gave rise to Sindbis virus and to the Sindbis-like parent of WEE. At some time after this divergence, a Sindbis-like virus belonging to the latter lineage was transferred to the Old World where it gave rise to Sindbis viruses distributed throughout the Old World, and in a separate event a Sindbis-like virus belonging to the same lineage underwent recombination with EEE to give rise to WEE.

Aura alphavirus subgenomic RNA is packaged into virions of two sizes.

The alphavirus genome is 11.8 kb in size. During infection, a 4.2-kb subgenomic RNA is also produced. Most alphaviruses package only the genomic RNA into virions, which are enveloped particles with icosahedral symmetry, having a triangulation number (T) = 4. Aura virus, however, packages both the genomic RNA and the subgenomic RNA into virions. The genomic RNA is primarily packaged into a virion that has a diameter of 72 nm and which appears to be identical to the virions produced by other alphaviruses. The subgenomic RNA is packaged into two major, regular particles with diameters of 72 and 62 nm. The 72-nm-diameter particle appears to be identical in construction to virions containing genomic RNA. The 62-nm-diameter particle probably has T = 3. The large and small Aura virions can be partially separated in sucrose gradients. In addition to these two major classes of particles, there are other particles produced that appear to arise from abortive assembly. From these results and from previous studies of alphavirus assembly, we suggest that during assembly of alphavirus nucleocapsids in the infected cell there is a specific initiation event followed by recruitment of additional capsid subunits into the complex, that the triangulation number of the complex is not predetermined but depends upon the size of the RNA and interactions that occur during assembly, and that budding of assembled nucleocapsids results in the acquisition of an envelope containing glycoproteins arranged in a manner determined by the nucleocapsid.

Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis.

Proteolytic processing of the Sindbis virus non-structural polyproteins (P123 and P1234) and synthesis of minus- and plus-strand RNAs are highly regulated during virus infection. Although their precise roles have not been defined, these polyproteins, processing intermediates or mature cleavage products (nsP1-4) are believed to be essential components of viral replication and transcription complexes. In this study, we have shown that nsP4 can function as the polymerase for both minus- and plus-strand RNA synthesis. Mutations inactivating the nsP2 proteinase, resulting in uncleaved P123, led to enhanced accumulation of minus-strand RNAs and reduced accumulation of genomic and subgenomic plus-strand RNAs. In contrast, no RNA synthesis was observed with a mutation which increased the efficiency of P123 processing. Inclusion of this mutation in a P123 polyprotein with cleavage sites 1/2 and 2/3 blocked allowed synthesis of both minus- and plus-strand RNAs. We conclude that nsP4 and uncleaved P123 normally function as the minus-strand replication complex, and propose that processing of P123 switches the template preference of the complex to minus-strands, resulting in efficient synthesis of plus-strand genomic and subgenomic RNAs and shut-off of minus-strand RNA synthesis.

Subgenomic mRNA of Aura alphavirus is packaged into virions.

Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.

Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus.

The polyprotein of classical swine fever virus starts with the nonstructural protein p23, which is followed by the nucleocapsid protein p14. Proteolytic cleavage between p23 and p14 was demonstrated in a cell-free transcription-translation system. Successive truncation of the cDNA used for the transcription indicated that the proteolytic activity responsible for the cleavage between p23 and p14 resides within p23. In order to determine the cleavage site between these two proteins, the respective genomic regions were expressed in two different expression systems. N-terminal sequencing of the resulting p14-related proteins revealed that cleavage occurs between Cys-168 and Ser-169. Comparison of the sequence around the cleavage site with sequences of other pestiviruses suggests a conserved processing site between similar proteins.

Processing of the envelope glycoproteins of pestiviruses.

The genomic RNA of pestiviruses is translated into a large polyprotein that is cleaved into a number of proteins. The structural proteins are N terminal in this polyprotein and include three glycoproteins called E0, E1, and E2 on the basis of the order in which they appear in the polyprotein. Using pulse-chase experiments, we show that a pestiviral glycoprotein precursor, E012, is formed that is processed into E0, E1, and E2 in an ordered fashion. Processing is initiated by a nascent cleavage between the capsid and the translocated E012 followed by cleavage at the C terminus of E2. E012 is then rapidly cleaved to form E01 and E2. After E2 is released from the precursor, E01 is processed into E0 and E1. To identify the sites of cleavage, the N termini of the glycoproteins of the pestivirus classical swine fever virus (formerly termed hog cholera virus) were sequenced after expression in the vaccinia virus system. The N termini are Glu-268 for E0 (gp44/48), Leu-495 for E1 (gp33) and Arg-690 for E2 (gp55). The sequences around the cleavage sites capsid/E0 and E1/E2 conform to the rules known for cellular signal proteases, as does the sequence at the presumed C terminus of E2. The sequence upstream of the E0/E1 cleavage site also shows sequence characteristics of signalase processing sites but lacks the typical hydrophobic signal peptide; this cleavage site has characteristics in common with a site in flaviviruses that is also cleaved in a delayed fashion. The absence of any membrane-spanning region results in the shedding of E0 by infected cells, and E0 can be detected in the virus-free supernatant. Comparison of the sequences around the cleavage sites of pestiviruses suggests a general processing scheme for the structural glycoproteins. Comparison of the pesti- and flaviviral structural glycoproteins suggests analogies between E012 and prM-E.

Molecular characterization of positive-strand RNA viruses: pestiviruses and the porcine reproductive and respiratory syndrome virus (PRRSV).

Molecular characterization has become an important tool for the analysis of viruses including their classification. The manuscript focuses on the molecular analysis of two members of the genus pestivirus (hog cholera virus, HCV and bovine viral diarrhea virus, BVDV) and of the recently discovered porcine reproductive and respiratory syndrome virus (PRRSV). The first protein encoded within the single large pestivirus ORF is a nonstructural protein with autoproteolytic activity. The cleavage site between the protease and the capsid protein p14 has been predicted previously, but recent experimental data indicate that processing occurs at a different site. The capsid protein is followed by a putative internal signal sequence and three glycoproteins which are part of the virion envelope. According to a new proposal for the nomenclature of the structural proteins of pestiviruses they are termed C, E0, E1 and E2. The genomes of BVDV pairs isolated from animals which came down with mucosal disease were analyzed. The genomes from cytopathogenic (cp) BVD viruses may contain insertions highly homologous to cellular sequences. In addition, cp BVDV may differ from its non cytopathogenic (noncp) counterpart by mere rearrangement of viral sequences. The disease PRRS, which emerged a few years ago, is caused by a single strand RNA virus; the viral genome is of positive polarity and has a size of 15 kb. Data concerning morphology, morphogenesis and virion composition suggested already that PRRSV belongs to a group of so-called arteriviruses which comprises equine arteritis virus (EAV), lactate dehydrogenase elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). This conclusion has now been confirmed by analysis of genome organization, gene expression strategy and by comparison of deduced protein sequences.

Proteins encoded in the 5' region of the pestivirus genome--considerations concerning taxonomy.

The first protein encoded within the pestivirus open reading frame is a nonstructural protein which removes itself from the polyprotein by autoproteolytic cleavage. The following nucleocapsid protein ends just before a putative signal sequence preceding three glycosylated proteins. All three glycoproteins are part of the viral envelope and exist in the form of disulfide-linked dimers. Pestiviruses have recently been reclassified as members of the family Flaviviridae which now comprises three genera, namely flavivirus, hepatitis C virus group and pestivirus. All members of the family have certain characteristics in common like the overall genome organization and the strategy of gene expression. Major differences exist, however, between the genera; the most obvious ones concern proteins encoded in the 5' region of the respective genomes.

Sindbis virus RNA polymerase is degraded by the N-end rule pathway.

Upon infection of animal cells by Sindbis virus, four nonstructural (ns) proteins, termed nsP1-4 in order from 5' to 3' in the genome, are produced by posttranslational cleavage of a polyprotein. nsP4 is believed to function as the viral RNA polymerase and is short-lived in infected cells. We show here that nsP4 produced in reticulocyte lysates is degraded by the N-end rule pathway, one ubiquitin-dependent proteolytic pathway. When the N-terminal residue of nsP4 is changed by mutagenesis, the metabolic stabilities of the mutant nsP4s follow the N-end rule, in that the half-life of nsP4 bearing different N-terminal residues decreases in the order Met greater than Ala greater than Tyr greater than or equal to Phe greater than Agr. Addition of dipeptides Tyr-Ala, Trp-Ala, or Phe-Ala to the translation mixture inhibits degradation of Tyr-nsP4 and Phe-nsP4, but not of Arg-nsP4. Conversely, dipeptides His-Ala, Arg-Ala, and Lys-Ala inhibit the degradation of Arg-nsP4 but not of Tyr-nsP4 or Phe-nsP4. We found that there is no lysine in the first 43 residues of nsP4 that is required for its degradation, indicating that a more distal lysine functions as the ubiquitin acceptor. Strict control of nsP4 concentration appears to be an important aspect of the virus life cycle, since the concentration of nsP4 in infected cells is regulated at three levels: translation of nsP4 requires read-through of an opal termination codon such that it is underproduced; differential processing by the virus-encoded proteinase results in temporal regulation of nsP4; and nsP4 itself is a short-lived protein degraded by the ubiquitin-dependent N-end rule pathway.

Hog cholera virus: molecular composition of virions from a pestivirus.

Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.

Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity.

A cDNA fragment covering the genomic region that encodes the structural proteins of hog cholera virus (HCV) was inserted into the tk gene of vaccinia virus. Expression studies with vaccinia virus/HCV recombinants led to identification of HCV-specific proteins. The putative HCV core protein p23 was demonstrated for the first time by using an antiserum against a bacterial fusion protein. The glycoproteins expressed by vaccinia virus/HCV recombinant migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. A disulfide-linked heterodimer between gp55 and gp33 previously detected in HCV-infected cells was also demonstrated after infection with the recombinant virus. The vaccinia virus system allowed us to identify, in addition to the heterodimer, a disulfide-linked homodimer of HCV gp55. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.

Molecular characterization of hog cholera virus.

An efficient tissue culture system was established which allowed to obtain substantial quantities of hog cholera virus (HCV) from the cell free tissue culture supernatant. After preparation of viral RNA and cDNA synthesis, the complete HCV genome was cloned and sequenced. Comparison with published BVDV sequences revealed a surprisingly high homology between HCV and BVDV at both the nucleotide and the amino acid level. In addition host cellular sequences were identified in BVDV genomes. The genomic localization of HCV glycoproteins was determined by the use of sequence specific antisera directed against bacterial fusion proteins. The order on the HCV genome was determined as follows: N-gp44/48-gp33-gp55-C. HCV gp33 and HCV gp55 were shown to be intracellularly linked by disulfide bridges. A cDNA fragment covering the genomic region that encodes the structural proteins of HCV was inserted into a vaccinia recombination vector. Expression studies with vaccinia/HCV recombinants led to identification of HCV specific glycoproteins which migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.

Insertion of cellular sequences in the genome of bovine viral diarrhea virus.

The genomic sequences of four pestiviruses, two BVDV strains (Osloss and NADL, both of which are cytopathogenic) and two HCV strains, were analyzed. Comparative studies revealed the presence of small insertions of cellular sequences in the genomes of both BVDV strains; the insertions are located in a region coding for a nonstructural protein. Such insertions are not present in the HCV sequences. The insertion identified in BVDV Osloss encodes a complete ubiquitin-like element. The sequence inserted in the BVDV NADL genome shows no homology to a ubiquitin gene but is almost identical with another bovine mRNA sequence. Molecular characterization of a BVDV "pair", isolated from an animal with mucosal disease, led to the detection of a ubiquitin-like sequence in the genome of the cytopathogenic strain, but not of the noncytopathogenic strain. It is proposed that recombination between viral and cellular RNA leads to formation of cpBVDV genomes. This hypothesis has direct implications for the pathogenesis of mucosal disease.

Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer.

Neutralizing monoclonal antibodies directed against hog cholera virus (HCV) precipitated two HCV-encoded glycoproteins, HCV gp55 and HCV gp33. Immunoassay with bacterial fusion proteins and Western immunoblotting with extracts from infected cells revealed that the antibodies recognized only HCV gp55. Coprecipitation of HCV gp33 was shown to be due to intermolecular disulfide bridges. One of the antibodies also reacted with the major glycoprotein of another pestivirus, bovine viral diarrhea virus (BVDV). The analogous BVDV glycoproteins exhibited a distribution of cysteine residues which was almost identical to that of HCV gp55 and gp33. The two BVDV glycoproteins were also linked by disulfide bridges.

Genomic localization of hog cholera virus glycoproteins.

A polyspecific antiserum has been used to identify four different glycoproteins in hog cholera virus (HCV)-infected cells termed gp55, gp48, gp44, and gp33 (Rümenapf et al, 1989, Virology 171, 18-27). Fusion proteins containing parts of the putative HCV-encoded glycoproteins were expressed in bacteria and served for the preparation of specific antibodies. These were used in radioimmunoprecipitation assays which revealed that gp48 and gp44 most likely share a common protein backbone. The order of the glycoproteins on the HCV genome was determined as follows: NH2-gp44/gp48-gp33-gp55-COOH.