INTRODUCTION

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The virus known as equine morbillivirus (EMV) first appeared in Hendra, a suburb of Brisbane, Australia, in September 1994 and was responsible for a brief but dramatic respiratory-disease outbreak, which resulted in the deaths of 14 horses and a horse trainer (29, 38). Over 1 year later, the virus was shown to be the cause of encephalitis and death of a horse breeder in Mackay, approximately 800 km north of Brisbane (31). The horse breeder probably became infected with EMV the previous year by assisting in the autopsies of horses which died with severe respiratory disease and were subsequently shown to have been infected with EMV (16). No direct link has been made between the two disease outbreaks (35). The recent finding that some 15% of Australian fruit bats (genus Pteropus), commonly known as flying foxes, have antibodies to EMV and the isolation of EMV-like viruses from bat uterine fluids suggest that fruit bats are a natural host for the virus (13, 30).

Virological and morphological analyses and sequence determinations of the M and F genes and part of the P gene indicated that the virus responsible for the original outbreak belonged to the family Paramyxoviridae (12, 17, 29). Sequence comparisons indicated that although homologies with other viruses were limited, the virus was more closely related to members of the Morbillivirus genus than to other Paramyxoviridae genera. Members of the Paramyxoviridae family contain predominantly monocistronic genes which are initiated at a single start codon and produce a single primary translation product. The P/V/C gene is a notable exception because it can produce multiple translation products both by using a series of initiation codons and overlapping reading frames (ORFs) and by an unusual cotranscriptional insertion of nontemplated nucleotides (6, 23). In addition to the major P protein, members of the Paramyxovirus and Morbillivirus genera produce a small basic (SB) protein (C) and a V protein, which shares the N-terminal region of P; they may generate other translation products, such as D and W proteins (23, 33). In members of the Paramyxovirus and Morbillivirus genera, as exemplified by Sendai virus (SeV) and measles virus (MeV), respectively, the primary mRNA transcript encodes the P protein and a single G insertion generates the V mRNA (2, 44). In contrast, members of the Rubulavirus genus do not produce a C protein and the unedited transcript of the gene codes for the V protein. A nontranscriptional insertion of two G residues generates the P protein mRNA (23).

Here we report the cloning and characterization of the P/V/C gene and the P protein of the new member of the Paramyxoviridae family known as EMV. The P protein is significantly larger than are other P proteins within the family, whereas the C protein is the smallest Paramyxoviridae C protein sequenced so far. Although the overall coding strategy for the P/V/C gene is similar to those of SeV and MeV, i.e., the primary transcript and one-G-insertion mRNA code for P and V proteins, respectively, there are features unique to this virus, such as the presence of an ORF between the C and V ORFs and an extended 3' untranslated tail in the P gene mRNA. These novel features and the lack of extensive serological cross-reactivities with members of the Morbillivirus genus (29) indicate that the provisional name of the virus, EMV, is inappropriate. Murray et al. (30) have suggested that the virus be called Hendra virus (HeV), after the Brisbane suburb from which it was first isolated. The length of the virus genome and the presence of long untranslated sequences in N, M, F, and G mRNAs (unpublished data) distinguish HeV from other Paramyxoviridae viruses and reinforce the need not only to adopt a more appropriate name for the virus but also to consider whether it should be classified in a new genus within the Paramyxovirinae subfamily (30).

	MATERIALS AND METHODS

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Cells and virus. Vero cells were grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum. HeV was isolated and plaque purified as previously described (17, 29). Stock preparations (titer, 2 × 10⁷ to 4 × 10⁷ 50% tissue culture infective doses [TCID₅₀]/ml) were generated by infection at a multiplicity of infection of 10⁴ TCID₅₀/cell. Virus was grown under biohazard level 4 conditions, in which laboratory workers wear positive-pressure encapsulating suits supplied with breathing air.

Virus purification. Vero cells were infected at a multiplicity of infection of 10² TCID₅₀/cell and incubated in Eagle's minimal essential medium containing 1% fetal calf serum at 37°C. At 48 h postinfection, culture medium was removed and clarified by centrifugation at 10,000 × g for 10 min in a type 19 rotor (Beckman). Virus was pelleted by centrifugation at 150,000 × g for 20 min in a type 55.2 rotor, resuspended in TNE (10 mM Tris, 100 mM NaCl, 1.5 mM EDTA [pH 7.4]) at 4°C overnight, layered onto linear 15 to 50% sucrose gradients in TNE, and centrifuged at 200,000 × g in an SW41 rotor for 20 min at 10°C. The virus band was removed, diluted in TNE, and pelleted at 200,000 × g in an SW41 rotor for 20 min at 10°C. The pellet was resuspended in TNE and used as a source of genomic RNA or ribonucleoprotein or inactivated by heating in 2% sodium dodecyl sulfate (SDS) at 100°C for 2 min prior to removal from the biohazard level 4 laboratory.

Construction of a cDNA library in the pZEro-1 cloning system. RNA was extracted from SDS-treated virus by standard methods, and 2 to 4 µg was used for cDNA synthesis with a TimeSaver cDNA synthesis kit from Pharmacia Biotech. Random hexamer primers (0.037 µg) were used for the synthesis of relatively long cDNA. Double-stranded cDNA with EcoRI adapters at both ends was ligated with EcoRI-digested vector pZEro-1 (Invitrogen, San Diego, Calif.), followed by electroporation into Escherichia coli host strain TOP10F' {F' [lacI^q Tn10 (Tet^r)] mcrA (mrr-hsdRMS-mcrBC) lacZM15 lacX74 deoR recA1 araD139 (ara-leu)7697 galU galK rspL (Str^r) endA1 nupG} and selection for the growth of recombinants in the presence of zeomycin (1). PCR screening of approximately 100,000 recombinants in a primary library with primers which anneal to flanking regions of the cloning site in the vector revealed that 70 to 75% clones contained inserts in the range from 0.2 to 2.0 kb. Plasmid DNAs from these recombinants were isolated as follows. About 2 µg of purified plasmid DNA was digested in separate tubes with restriction enzymes PstI, SacI, and XbaI, which cut once in the vector multiple cloning site. Linearized plasmid DNA was purified from a 1% agarose-TAE (0.04 M Tris-acetate, 2 mM EDTA) gel with a GeneClean kit (Bio 101, San Diego, Calif.), and 4- to 7-kb fragments from the three digestion mixtures were combined, self-ligated, and reintroduced into TOP10F' by electroporation to form a secondary library. PCR screening indicated that about 90% of clones in the secondary library contained inserts ranging from 0.4 to 2 kb, with a majority around 0.7 to 1.2 kb.

Library screening and characterization of overlapping cDNA clones. The following two PCR approaches were used for the generation of specific gene probes to screen the cDNA library: (i) conventional PCR amplification with two specific primers that anneal to the ends of a known sequence and (ii) suppression PCR amplification with only one gene-specific primer (39). Approximately 1 µg of plasmid DNA purified from the primary library was digested with blunt-end-generating enzymes RsaI, HaeIII, and HincII, and fragments were ligated with 20 pmol of an adapter (top strand, 5' CTAAT ACGAC TCACT ATAGG GCTCG AGCGG CCGCC CGGGC AGGT-3'; bottom strand, 5'-P_i-ACCTG CCC-NH₂-3') to form a random adapter-anchored library to be used as template DNA in suppression PCR. One microliter of a 1:500 dilution of the ligation mixtures discussed above was used for PCR amplification with the following cycle parameters. For primary PCR, the first step consisted of 7 cycles at 94°C for 25 s and 72°C for 3 min and the second step consisted of 32 cycles at 94°C for 25 s and 67°C for 3 min, followed by an additional 7 min at 67°C after the final cycle. Similar conditions were used in secondary PCR, except that 5 and 20 cycles were used for the first and second steps, respectively. Primers AP1 (5'-GGATC CTAAT ACGAC TCACT ATAGG GC-3') and AP2 (5'-AATAG GGCTC GAGCG GC-3'), which anneal to the adapter sequence, were used in primary and secondary PCRs, respectively, together with a gene-specific primer that anneals to the end of a known cDNA sequence. PCR fragments (usually in the range of 150 to 300 bp) generated by either of these approaches were purified from a 2% agarose-TAE gel with a GeneClean kit before being labeled with [³²P]dATP by using a Ready-To-Go DNA labeling kit (Pharmacia Biotech). Colony hybridization was carried out with a colony/plaque screen hybridization transfer membrane (NEN Research Products, Du Pont, Boston, Mass.). From four rounds of screening by a genome walking strategy, a total of six overlapping cDNA clones, which covered the entire P gene, were isolated. Sequences from the overlapping clones were determined with universal and reverse sequencing primers, which annealed to the flanking regions of the pZEro-1 vector cloning sites, and gene-specific primers derived from information obtained by genome walking analyses. The nucleotide at each position was sequenced at least twice, either from two overlapping clones or from both strands. The complete cDNA sequence derived from overlapping cDNA clones was corroborated by direct sequencing of PCR fragments obtained from the viral genome by reverse transcription-PCR (RT-PCR) (see below). Except for two nucleotide changes (only one of which resulted in a change in the amino acid sequence), the genome-derived sequence was identical to that derived from individual cDNA clones.

Synthesis of cDNA by RT-PCR. Total nucleic acid was extracted from virus-infected Vero cells with TRIZOL reagent (Life Technologies) by a single-step isolation procedure (3). Virus-specific cDNAs were synthesized by using the Superscript preamplification system (Life Technologies) with gene-specific primers, followed by PCR amplifications with the following parameters: for primary PCR, denaturation at 94°C for 1 min, annealing at 50°C for 2 min, and elongation at 72°C for 2 min for 30 cycles, followed by an additional 7 min at 72°C after the final cycle; for secondary PCR, the same conditions as those used for primary PCR with internal primers.

Determination and analysis of nucleotide sequence. PCR products, derived from cloned cDNA or genomic RNA by RT-PCR, were sequenced with Sequenase PCR sequencing kits (Amersham Life Science). Sequences were manipulated by using the Clone Manager program (version 4.01; S&E Software). Multiple-sequence alignments were done by using ALIGN Plus (version 3.0; S&E Software), CLUSTAL W (version 1.6) (42), and the SAM sequence alignment and modelling software system (22). Phylogenetic analyses were performed by maximum-parsimony (PROTPARS program) and distance matrix (PROTDIST and NEIGHBOR programs) methods in the PHYLIP package (10). Trees were generated from 100 random data sets by using the SEQBOOT program, and majority-rule bootstrapped trees were constructed by using the CONSENSE program (10). Only branches with greater than 50% bootstrap support were included.

In situ enzymatic digest and peptide mapping. The method of Moritz et al. (28) was adapted to obtain internal amino acid sequence data after enzymatic digestion of proteins on gels. Proteins of SDS-treated virus were separated by polyacrylamide gel electrophoresis (PAGE) on Novex precast gels. Samples in 2% SDS were reduced and incubated for 5 min at 100°C. Two 10% gels were prerun with electrophoresis buffer containing 15 mg of reduced glutathione (Sigma) per liter for 90 min at 3 mA. Samples containing approximately 200 µg of total protein were loaded on each gel and separated at 100 V with glutathione-free electrophoresis buffer. Gels were stained for 30 min in 0.1% Coomassie brilliant blue R-250 and destained in 5% acetic acid-20% methanol. P protein bands were excised from gels and kept in airtight containers at 20°C. Two additional pieces of each gel containing no apparent protein were used as negative controls. Gel fragments were cut into 2-mm-long pieces and washed in 2 M NH₄HCO₃-50% acetonitrile for 30 min at room temperature with occasional mixing. The washing step was repeated twice. Gel pieces were dried in a SpeedVac concentrator (Savant) for 30 min and rehydrated in 20 µl of digestion buffer (100 mM NH₄HCO₃, 50% acetonitrile) for 30 min at 37°C. Modified trypsin or endoproteinase Glu-C (sequencing grade; Boehringer Mannheim) was used at an enzyme/substrate ratio of 1:10 in digestion buffer. Gels were incubated overnight at 37°C in 150 µl of digestion buffer and pelleted in a microcentrifuge. Supernatants containing peptide fragments were retained. Residual gel pieces were washed with 200 µl of 1% trifluoracetic acid (TFA) at 37°C for 30 min, centrifuged, and washed again with 200 µl of 0.05% TFA-80% acetonitrile at 37°C for 30 min. Supernatants were combined, concentrated in a SpeedVac to approximately 50 µl, diluted to 100 µl with 0.05% TFA in water, and subjected to reverse-phase chromatography.

Reverse-phase chromatography. Chromatography of enzymatically digested protein samples was performed on a System Gold high-performance liquid chromatograph (Beckman) with diode array detectors (model 168; Beckman). Peptides were separated on a Vydac C₁₈ reverse-phase column (2.1 by 250 mm; Separations Group, Hysperia, Calif.) operated at 43°C and by using a binary gradient with 0.05% TFA as buffer A and 0.045% TFA-80% acetonitrile as buffer B. The gradient was developed as follows: 2 to 37.5% buffer B over 0 to 50 min, 37.5 to 75% buffer B over 50 to 75 min, and 75 to 100% buffer B over 75 to 85 min at a flow rate of 150 µl/min. About 40 fractions were collected manually and stored at 20°C prior to automated sequencing.

Edman degradation amino acid sequencing. Protein and peptides were subjected to automated (Edman degradation) sequence analysis (8) with vapur phase delivery of critical reagents (15) in an automated sequenator (model 470A; Applied Biosystems) in conjunction with a PTH amino acid separation system (model 120A PTH analyzer; Applied Biosystems).

SDS-PAGE, Western blotting, and enzyme-linked immunosorbent assay. Standard procedures were used for SDS-PAGE, Western blotting, and enzyme-linked, immunosorbent assay (45-47).

Nucleotide sequence accession number. The sequence reported here has been assigned GenBank accession no. AF010304.

	RESULTS

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Virus proteins. The proteins in purified HeV are shown in Fig. 1. Based on the presence of L, P, and N proteins in CsCl buoyant density-purified HeV ribonucleoprotein, the Triton X-100-mediated release of proteins G, F₀, F₁, and F₂ from purified virus, and the reactions of proteins on Western blots with antisera raised to bacterially expressed portions of individual proteins (unpublished data), the proteins were designated as indicated in Fig. 1. Like those of other Paramyxoviridae family members, the P protein of HeV is phosphorylated (data not shown). While the molecular masses of most proteins calculated on the basis of electrophoretic mobility were similar to those of proteins from other Paramyxoviridae viruses, the estimate of 98 kDa for the P protein was significantly larger than the masses of P proteins from other family members, such as human parainfluenza virus type 3 (hPIV3) (83 kDa) (48), MeV (70 kDa) (41), and mumps virus (68 kDa) (27). The amino acid sequences of many Paramyxoviridae P proteins have been predicted from nucleic acid sequence data, and P protein sizes range from 241 amino acids (aa) for pneumoviruses to 507 and 603 aa for parainfluenza viruses and morbilliviruses, respectively. To characterize the HeV P protein, the gene and protein were sequenced and compared with the P proteins of other Paramyxoviridae viruses.

View larger version (62K): [in this window] [in a new window]   FIG. 1.   Analysis of HeV structural proteins by PAGE. The molecular weights of Coomassie brilliant blue-stained marker and HeV proteins are indicated on the left and right, respectively.

Characteristics of the P/V/C gene and its coding capacity. The complete sequences of the P/V/C gene and its potential coding regions are presented in Fig. 2. The predicted P/V/C gene starts and stops with highly conserved transcription initiation and termination signals, respectively (Fig. 2). The trinucleotide intercistronic sequence CTT, conserved in Paramyxoviridae viruses, was observed both upstream and downstream of the P gene (not shown). As summarized in Table 1, the mRNA is 2,698 nucleotides in length and appears to be capable of coding for several proteins, as has previously been observed for the P/V/C genes of other Paramyxoviridae viruses (23).

View larger version (41K): [in this window] [in a new window]   View larger version (49K): [in this window] [in a new window]   FIG. 2.   DNA, editing site, and ORF sequences of the HeV P/V/C gene. DNA and deduced amino acid sequences are shown in the first and second lines, respectively, of each set. Gene initiation and termination signals are underlined. ORF designations consisting of the encoded proteins and reading frames are indicated in bold and are followed by the deduced amino acid sequences of the encoded proteins. The conserved editing site for the insertion of a single G residue is in bold.

Amino acid sequence analysis of the P protein. Given the very low levels of sequence homology observed between the P/V/C gene of HeV and those of other Paramyxoviridae viruses (see below), amino acid sequence determinations of peptides derived from the P protein were used to corroborate the amino acid sequence deduced from the P/V/C gene sequence. The HeV P protein separated on SDS-PAGE gels as a well-defined protein band (Fig. 1) that was excised from gels or from membranes after electrotransfer. Direct N-terminal amino acid sequencing of P protein immobilized on a polyvinylidene difluoride membrane gave no sequence data, indicating that the N terminus of the protein was chemically blocked. Similar results were obtained in experiments with aldehyde-free acetic acid used in staining procedures, indicating that the N terminus of the P protein may be modified. Trypsin and Glu-C endoproteinase were used to generate internal peptides from protein samples excised from polyacrylamide gels. As summarized in Table 2, the 71% of the internal amino acid sequence obtained from these peptides was found to be identical with that derived from DNA analysis. No sequence information could be obtained for the blocked N-terminal peptide (aa 1 to 22) or for three long peptides (aa 459 to 530, 540 to 553, and 558 to 587) associated with the protein's hydrophobic core. These peptides were not identified in proteolytic digests, as they were possibly not eluted from the reverse-phase column due to their hydrophobic natures.

Multiple-sequence alignments and phylogenetic analysis of P/V/C gene-encoded proteins. An alignment of the P/V/C gene-encoded proteins of HeV by using the ALIGN Plus, SAM, and CLUSTAL W programs indicated that HeV diverges significantly from other members of the Paramyxoviridae family. Rubulaviruses in particular are only distantly related, and even with the more closely related paramyxoviruses and morbilliviruses, the HeV P protein displays amino acid sequence identities of only 11 to 14 and 12 to 16%, respectively (Table 3). Low levels of homology are also characteristic of the HeV C protein, with amino acid sequence identities of 7 to 13 and 10 to 13% to C proteins from paramyxoviruses and morbilliviruses, respectively. These figures are substantially lower than those observed for P and C proteins of viruses within either genus. The amino acid sequence identities for P and C proteins in morbilliviruses are 44 to 48 and 37 to 42%, respectively. The corresponding figures for Paramyxovirus genus members are 23 to 53 and 36 to 69% (Table 3). In contrast to the P and C proteins, the HeV V-specific region shows 30 and 52% homologies with paramyxovirus and morbillivirus V-specific proteins, respectively. The latter figure lies within the range of 50 to 71% exhibited by morbilliviruses (Table 3). No significant sequence homology was detected between sequences in protein data banks and the putative small basic protein.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   Sequence alignment revealing regions of homology between HeV P and V proteins and cognate proteins of selected Paramyxovirinae viruses. (A) Alignment of the P protein from the insertion site to the carboxy terminus. Amino acids encoded by the conserved insertion sequence are indicated by asterisks. Amino acid numbers of the HeV P protein are indicated above the sequence. (B) V protein alignment. Insertions in one protein are indicated by dashes in the sequences of cognate proteins. Amino acid identities and conserved amino acid characteristics are indicated below sequences by asterisks and periods, respectively.

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Phylogenetic trees for the HeV P/V/C gene (A) and P (B), C (C), and V (D) proteins. Phylogenetic trees were produced from 100 random data sets generated by using the SEQBOOT program from PHYLIP (10) and are shown as majority-rule consensus, radial trees constructed by maximum-parsimony methods (PROTPARS and CONSENSE programs [10]). Only branches with >50% bootstrap support are indicated, and trees are unrooted with branch lengths not drawn to scale.

	DISCUSSION

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The P/V/C gene of HeV differs from other Paramyxovirinae genes in a number of ways. (i) The gene is much larger. (ii) The P protein is the largest described to date. (iii) HeV P mRNA has a much longer untranslated 3' tail. (iv) HeV C protein is smaller and less basic than cognate proteins in other Paramyxovirinae viruses. (v) All Paramyxovirinae viruses express at least two ORFs from their P/V/C genes. Only the P protein is expressed in all of the viruses studied; the C protein is absent in rubulaviruses, and some parainfluenza viruses do not produce the V protein. However, in addition to P, C, and V proteins, the P/V/C gene in HeV has the coding capacity for a putative SB protein.

The recognition that more than one protein is generated from a single mRNA in Paramyxovirinae viruses was an early example of multiple translational initiation, which led to modification of the scanning model for ribosomal initiation (20). In the modified model, scanning ribosomes can occasionally bypass 5'-proximal ATGs in an unfavorable context and initiate protein synthesis at downstream ATGs. According to Kozak, the optimal context for translation initiation has the consensus sequence GCCA(G)CCATGG, in which the A (sometimes G) residue at 3 and the G residue at +4 play the most important roles in modulating initiation efficiency. In the HeV P/V/C gene, we have found TGACAAATGG for ORF-P, TCAATGATGG for ORF-C, and ATTACTATGG for ORF-SB (Fig. 2). Since all three sequences have a G residue at +4, their initiation efficiencies are most likely determined by the residue at 3. Both ORF-C and ORF-SB have the optimal residue, A, at this position, whereas ORF-P has a nonpurine residue, C, making the ORF-P initiation site the weakest. This observation is consistent with the hypothesis that ORF-P contains a leaky initiation site for translation. For ORF-C, there are two tandem ATG codons at the beginning of the ORF; the second ATG was used in this comparison since it gives a better initiation context. ORF-C and ORF-SB have identical residues at 3 and +4. In addition, ORF-SB has a C residue at 2, whereas ORF-C has a C residue at 5. It is therefore difficult to rank the initiation environments for these two ORFs.

In relation to coding capacity for multiple proteins in different reading frames of the HeV P/V/C gene, it is interesting that the protein encoded in the +1 reading frame is very acidic (the P protein), those encoded in the +2 reading frame are basic (C and SB proteins), and the protein encoded in the +3 reading frame is close to neutral (V-specific protein).

Within the P/V/C genes of Paramyxovirinae viruses, the C-terminal domain of the P protein, i.e., the region after the mRNA editing site, is the most conserved. It is essential for genome replication (4), oligomerization of the P protein (14, 23), binding to the L protein (25), and association with the N protein both in nucleocapsids (36) and in unassembled N-P complexes (5). For most Paramyxovirinae viruses, this is also the V-specific coding region. In the case of the HeV P protein, the domain after the mRNA editing site also represents the region which displays the best, albeit low, homologies with the P proteins of morbilliviruses and paramyxoviruses. It is interesting that although the overall size of HeV P protein is much larger than its counterparts in other Paramyxovirinae viruses, this C-terminal domain (304 aa) is only slightly longer than those in other P proteins (253 to 277 aa).

The presence of an ORF coding for an SB protein between ORF-C and ORF-V is a unique feature of the HeV P/V/C gene. A similar SB protein, encoded in an overlapping reading frame within the P gene, has previously been identified in both the New Jersey and Indiana serotypes of VSV (21, 40). The expression of VSV SB in infected cells has previously been experimently demonstrated, and the protein does not appear to be associated with virions. Sequence analysis indicated that the SB protein is conserved in the Vesiculovirus genus of the Rhabdoviridae family but not in the Lyssavirus genus. It is interesting that both VSV and HeV SB proteins are 65 aa in length and contain eight arginine residues, which contribute to the high pIs of 11.0 and 11.9 for VSV SB and HeV SB, respectively. A similar SB-encoding ORF has also previously been found in the VP35 gene, the equivalent of the P gene, of Marburg virus in the Filoviridae family (9). If the HeV and Marburg virus SB proteins are synthesized in vivo, SB proteins may represent a class of molecules which are shared among the three families within the Mononegavirales order. Although SB proteins are not encoded by all viruses within a family and there seems to be no conservation of primary sequence, it is interesting that these P-overlapping molecules in members of the Mononegavirales order have very similar molecular sizes and characteristics. Spiropoulou and Nichol (40) used the letter C to designate the SB protein synthesized by VSV because its location at the N terminus of ORF-P and its overall basic nature resemble that of the C protein of a Paramyxovirinae virus. However, we believe that it may be more appropriate to name them SB proteins for two reasons. First, these proteins are much smaller, approximately one-third of the size of C proteins identified in Paramyxovirinae viruses. Second, we have identified in HeV not only a C protein but also a coding region that is potentially capable of generating an SB protein which is equivalent in size and with characteristics similar to the SB proteins identified in vesiculoviruses and Marburg virus.

The P genes of almost all of the viruses in the order Mononegavirales have the capacity to code for more than one protein either by multiple translational initiation or mRNA editing, but proteins such as V and C are not found in all viruses and may therefore be described as accessory proteins. Among the various proteins expressed from the P/V/C gene, the P protein is the only one known to be essential for virus replication and synthesized by all viruses in the family. The function of the C protein is not clear, and this protein is not present in rubulaviruses. The V protein is interesting for two reasons. First, it is cysteine rich, contains a zinc finger, and has previously been shown to bind zinc (24, 32). Second, V proteins are the most conserved proteins encoded by the P/V/C gene, arguing for their importance in virus evolution. Until recently, the V protein was regarded as nonessential, at least in tissue culture cells (7, 37). In 1997, Kato et al. (18, 19) demonstrated that SeV V protein codes for as a function that is required for viral replication and expression of pathogenesis in vivo. Whether the in vivo function of the V protein is unique to SeV remains to be seen because the closely related virus hPIV1 lacks the capacity to code for a V protein (26, 34) and hPIV3 does not seem to express a V protein, although the coding sequence is present in the P/V/C gene (11). In addition to V and C proteins, HeV may code for a third accessory protein, SB. It is tempting to speculate that the large P gene of HeV and its capacity to code for up to three unrelated, accessory proteins may be responsible, at least partially, for the ability of the virus to replicate in species as diverse as horses, humans, cats, rabbits, and fruit bats (49, 51) and in a wide range of cultured cells (29). HeV is pathogenic in humans, horses, cats, and guinea pigs (50).

The unusually large size of the HeV P protein, the nonconserved nature of its N-terminal domain, and the fact that the HeV P/V/C gene and its products have sequences that diverge significantly from those of other members of the Paramyxoviridae make the construction of multiple-sequence alignments difficult to interpret. Attempts were made to overcome this by (i) validating the phylogenetic groupings with bootstrap resampling to give confidence limits to the trees and (ii) using two methods of phylogenetic analysis, distance matrix-neighbor-joining and maximum-parsimony methods. The trees produced were consistent with those generated for the more conserved M protein (12) and gave bootstrap values of >50%. The results show clearly that HeV cannot be classified as either a paramyxovirus or a morbillivirus; it forms a distinct branch in all phylogenetic trees, suggesting that HeV should be classified in a new genus within the Paramyxovirinae subfamily (30).