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The nucleotide sequences at the 3'-termini of human parainfluenza virus 3 (HPIV3) genomic RNAs recovered from two lines of persistently infected LLC-MK2 cells were determined following PCR amplification. After 29 months of persistence the 3'-end of the HPIV3 genome was found to be highly mutated. Interestingly, the only types of nucleotide changes observed were U to C and A to G transitions. Both U to C and A to G transitions were present on individual RNA molecules. The data indicate that biased hypermutational activity leading to U To C and A to G mutations operates in cultured cells during persistent HPIV3 infections.

cDNA clones representing the fusion (F) gene of human parainfluenza virus type 2 (PIV-2) were isolated from cDNA libraries constructed from virus-specific mRNA and genomic RNA, and the complete nucleotide sequence of the F gene was determined. The F gene is 1854 nucleotides long and encodes one long open reading frame of 551 amino acids. The cleavage site for activation of the precursor Fo protein is Thr-Arg-Gln-Lys-Arg. The F gene of PIV-2 is most closely related to those of simian virus 5 (SV5) and mumps virus (MuV). Interestingly, although the HN glycoprotein of PIV-2 shows no relatedness to the HA glycoprotein of measles virus (MV), a distinct homology is found in the F proteins of PIV-2 and MV. As concerns F proteins, paramyxoviruses can be divided into two subgroups; that is, PIV-2, SV5, and MuV belong to one group, and HPIV-1, SV, and PIV-3 belong to the other group. Newcastle disease virus (NDV) and MV are intermediate. Coding regions for small hydrophobic (SH) proteins have been found between the HN and F genes of SV5 and MuV, which are the viruses most closely related to PIV-2. However, such a gene could not be detected in two different strains of PIV-2.

We cloned and sequenced the cDNAs against genomic RNAs and mRNAs for phosphoproteins (Ps) of human parainfluenza virus types 4A (PIV-4A) and 4B (PIV-4B). The PIV-4A and -4B P genes were 1535 nucleotides including poly(A) tract and were found to have two small open reading frames, neither of which was apparently large enough to encode the P protein. A cluster of G residues was found in genomic RNA and the number of G residues was 6 in both PIV-4A and -4B. However, the number of G residues at the corresponding site in the mRNAs to the genomic RNA was not constant. Three different mRNA cDNA clones were obtained; the first type of mRNA encodes a larger (P) protein of 399 amino acids, the second type encodes V protein of 229 or 230 amino acids, and the third type encodes the smallest protein (156 amino acids). Comparisons on the nucleotide and the amino acid sequences P and V proteins between these two subtypes revealed extensive homologies. However, these homology degrees are lower than that of NP protein. The C-terminal regions of the P and V proteins of PIV-4s could be aligned with all other Paramyxoviruses, PIV-2, mumps virus (MuV), simian virus 5 (SV 5), Newcastle disease virus (NDV), measles virus (MV), canine distemper virus (CDV), Sendai virus (SV), and PIV-3. On the other hand, the P-V common (N-terminal) regions showed no homology with MV, CDV, SV, and PIV-3. Seven phylogenetic trees of Paramyxoviruses were constructed from the entire and partial regions of P and V proteins.

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.

Variations in epitopes on structural proteins of four isolates of parainfluenza virus type 4 (PIV-4) and the Mr of polypeptides in these isolates were determined by radioimmune precipitation assay with monoclonal antibodies to parainfluenza virus type 4A (PIV-4A) and type 4B (PIV-4B). Three isolates antigenically resembled the prototype PIV-4A and the sizes of their structural proteins were 72K (HN protein), 61K (F0 protein), 61K (NP protein) and 40K (M protein). However, one virus isolate showed marked antigenic differences from both the 4A and 4B prototype viruses, particularly with regard to properties of the HN and F proteins. In addition, both the NP and F0 proteins of this isolate had a slightly increased Mr of 63K.

We cloned and sequenced the cDNAs against genomic RNA and mRNA for phosphoprotein (P) of human parainfluenza type 2 virus (PIV-2). cDNA clone from genomic RNA was 1439 nucleotides in length excluding poly(A) and was found to have two small open reading frames encoding proteins of 233 and 249 amino acids. Two different mRNA cDNA clones were obtained; that is, one mRNA contained a smaller reading frame coding 225 amino acids, V protein, and the other mRNA contained a larger reading frame coding 395 amino acids, P protein. Both mRNAs had G cluster in coding frame. The former mRNA contained seven G residues, and two extra G residues were inserted in the latter mRNA. Ten cDNA clones from the genomic RNA were identical and were composed of seven G residues, indicating that genomes analyzed here were a homogeneous population. Therefore, V protein is encoded by faithfully copied mRNA and P protein is translated from mRNA in which two additional G residues are nontemplately inserted immediately after seven genomically encoded G residues. The V and P proteins are amino coterminal proteins and have different C termini. The C terminus of V protein is cysteine-rich and bears some resemblance to metal-binding protein of the zinc finger-type motif. P protein sequence of PIV-2 showed high homologies with SV 5 (40.4%) and mumps virus (35.5%), and a moderate homology with Newcastle disease virus (20.6%). On the other hand, very little homology was found between PIV-2 and other paramyxoviruses including Sendai virus, PIV-3, and measles virus. The cysteine-rich region in V protein was found to be highly conserved in PIV-2, SV 5, and measles virus, suggesting that V protein of paramyxoviruses plays important roles in transcription and/or replication. The predicted cysteine-rich V protein was detected in virus-infected cells using antiserum directed against an oligopeptide specific for the predicted V polypeptide.

The nucleotide sequence of the "P/V gene" of parainfluenza virus type 2 is presented. To determine the nature of any nontemplated additions of nucleotides that may arise during the synthesis of mRNA from this gene the polymerase chain reaction was used to amplify specific sequences of both genomic RNA and mRNA. These results demonstrated that the V protein is encoded by the genome while insertion of two nontemplated G residues are required to synthesize P mRNA. The predicted P protein has 44% identical amino acid homology with that of simian virus 5 and 37% with mumps virus; 25% of the amino acids is conserved between all three viruses.

Three cell lines persistently infected with human parainfluenza virus type 3 were characterized on a molecular level in this study. All six structural protein genes were transcribed into monocistronic RNAs in the persistently infected cells. In both acutely and persistently infected cells, polycistronic transcripts were abundant, although the ratio of polycistronic to monocistronic transcripts was reduced in the persistently infected cells. Each of the persistently infected cell lines contained a distinct subgenomic RNA species. The subgenomic RNAs were present in purified nucleocapsid cores, indicating that they represent viral genome RNA, were far more abundant than full-length RNA, and were stably maintained through at least 36 cell passages. Nucleotide sequence analysis of the subgenomic RNAs from two of the persistently infected cell lines revealed that the 5' ends are identical to that of the standard genome. Hybridization experiments with oligonucleotide probes showed that both fragments retain sequences from the 5' end of the standard genome and contain approximately 1,200 nucleotides (cell line 1) and 1,500 nucleotides (cell line 2) of the polymerase gene sequence. The demonstration of several alterations in viral gene expression in persistently infected cells offers insight into the factors associated with persistence of parainfluenza virus 3.

A cDNA library was constructed in lambda gt10 using mRNA purified from cells infected with parainfluenza virus type 2 (PIV2). Virus-specific clones were identified by screening the library with 32P-labelled cDNA probes made from randomly primed vRNA. Clones containing the haemagglutinin-neuraminidase (HN) gene were identified by sequence comparisons with known parainfluenza virus HN gene sequences. The largest HN clone isolated had a nucleic acid sequence of 2065 bp with a single long open reading frame encoding a protein of 571 amino acids. The HN protein has nine predicted glycosylation sites and an amino-terminal membrane-spanning region. The PIV2 HN protein shares 43% amino acid identity with the HN protein of simian virus 5 and 40% with mumps virus, 30% of the amino acids being common to all three viruses.

We cloned the cDNA of human parainfluenza type 4A virus (PIV-4A) HN gene by reverse-transcription of virus-specific mRNAs and genomic RNA, and determined the complete nucleotide sequence of the HN gene. The predicted HN protein sequence of PIV-4A showed significant relatedness with those of other paramyxoviruses, SV5, NDV, MuV, PIV-3, BPIV-3, indicating a common ancestor. The homologies between the viruses suggested that PIV-4A is more closely related to NDV, SV5, and MuV than to the parainfluenza viruses, PIV-3, bovine parainfluenza type 3 virus (BPIV-3), and Sendai virus (SV). Sixty amino acids were commonly conserved among the viruses, other than PIV-4A. Two of these amino acids were substituted in PIV-4A HN and are predicted to be located near the active site of the neuraminidase. The analysis of neuraminidase of PIV-4 revealed that the activity is hardly detectable, suggesting the significant effect of the substituted amino acid sites on neuraminidase activity.

Many human parainfluenza type 3 virus (PIV3) strains isolated from children with respiratory illness are resistant to neutralization by monoclonal antibodies (MAbs) which recognize epitopes in antigenic site A or B of the fusion (F) protein of the prototype 1957 PIV3 strain. The F protein genes of seven PIV3 clinical isolates were sequenced to determine whether their neutralization-resistant phenotypes were associated with specific differences in amino acids which are recognized by neutralizing MAbs. Several clinical strains which were resistant to neutralization by site A or B MAbs had amino acid differences at residues 398 or 73, respectively. These specific changes undoubtedly account for the neutralization-resistant phenotype of these isolates, since identical substitutions at residues 398 or 73 in MAb-selected escape mutants confer resistance to neutralization by site A or B MAbs. The existence of identical changes in naturally occurring and MAb-selected neutralization-resistant PIV3 strains raises the possibility that antigenically different strains may arise by immune selection during replication in partially immune children. Three of the seven clinical strains examined had differences in their F protein cleavage site sequence. Whereas the prototype PIV3 strain has the cleavage site sequence Arg-Thr-Lys-Arg, one clinical isolate had the sequence Arg-Thr-Arg-Arg and two isolates had the sequence Arg-Thr-Glu-Arg. The different cleavage site sequences of these viruses did not affect their level of replication in either continuous simian or bovine kidney cell monolayers (in the presence or absence of exogenous trypsin or plasmin) or in the upper or lower respiratory tract of rhesus monkeys. We conclude that two nonconsecutive basic residues within the F protein cleavage site are sufficient for efficient replication of human PIV3 in primates.

A cell extract derived from human parainfluenza virus type 3-infected human lung carcinoma (HLC) cells synthesized mRNA in vitro. Under optimal conditions, the extract was able to support transcription of all virus-encoded genes as determined by hybridization analyses. The RNA products contained full-length poly(A)-containing mRNA species similar to those observed in acutely infected cells. Further purification of the viral nucleocapsids from the infected HLC cell extract resulted in total loss of the capacity of the extract to synthesize mRNA in vitro. However, the addition of cytoplasmic extracts from uninfected HLC cells to the nucleocapsid preparations restored transcription to levels observed in the infected cell lysates, indicating requirement of a host factor(s) in the human parainfluenza virus type 3 transcription process. In distinction to the abundant transcription observed in the cell extract from HLC cells, cell extract prepared from CV-1 cells failed to support transcription in vitro. High levels of RNase activity in the cell extract from CV-1 cells appears to be the principal reason for this difference.

The nucleotide sequences of the cDNA copies of the mRNA coding for the nucleocapsid proteins (NPs) of human parainfluenza viruses type 4A (PIV-4A) and type 4B (PIV-4B) were determined. The copy of PIV-4A NP mRNA contained 1885 nucleotides encoding a protein with a calculated molecular weight of 62,561. The same number of amino acids with a similar molecular weight (62,425) were predicted for the PIV-4B NP protein. Comparisons on the nucleotide sequence and the amino acid sequence of NP protein between these two subtypes revealed extensive homologies in the nucleotide sequence (87%) and in the amino acid sequence (93%). Furthermore, a conserved region with about 100 amino acids was observed between PIV-4s and other paramyxoviruses, Newcastle disease virus (NDV), Sendai virus, mumps virus (MuV), PIV-3, BPIV-3, measles virus (MV), and canine distemper virus (CDV), indicating a common ancestor for these nine viruses. Our data also indicated that the PIV-4 NP proteins were more closely related to MuV and NDV than to other parainfluenza viruses, PIV-3, BPIV-3, and Sendai virus. Interestingly, the NP protein homology between PIV-4s and the morbillivirus group, MV and CDV, was slightly higher than that between PIV-4s and the parainfluenza viruses, PIV-3, BPIV-3, and Sendai virus.

The nucleotide sequence of the hemagglutinin-neuraminidase (HN) gene of human parainfluenza type 2 virus (PIV-2) was determined. The PIV-2 HN gene was 2112 nucleotides excluding poly(A) tail. There was a single large open reading frame in the mRNA which encoded a protein of 571 amino acids with a calculated molecular weight of 63,262. Analysis of the deduced amino acid sequence revealed that there were fourteen potential glycosylation sites and a major hydrophobic region near the N-terminus, which would anchor the protein in the viral membrane. Comparisons of the HN protein sequences of PIV-2 with those of Simian virus 5 (SV5), Sendai virus (SV, parainfluenza virus type 1), human parainfluenza virus type 3 (PIV-3), type 4 (PIV-4), bovine parainfluenza virus type 3 (BPIV-3), mumps virus (MuV), and Newcastle disease virus (NDV) showed definite amino acid sequence relatedness, indicating a common ancestor for these viruses. Furthermore, statistical analysis of the protein sequences suggested a possible evolutionary relatedness among the paramyxoviruses. This is the first time that a phylogenetic tree has been constructed for all the parainfluenza viruses and mumps virus which are infectious to humans. In addition, amino acid sequences involved in hemagglutinating and neuraminidase activities of paramyxovirus were discussed.

The sequence of the genes encoding the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of the human parainfluenza 3 virus was determined by molecular cloning. The genes were cloned by primer extension using genomic 50 S RNA as the template. A series of four overlapping clones was generated from the 3' end of the fusion gene which extended across the gene end and intergenic boundaries of the F-HN and HN-L genes. The F gene extends 1851 nucleotides (inclusive of the putative transcription initiation and polyadenylation signals) and encodes a protein consisting of 539 amino acids (mol wt 60,067). This protein contains four potential sites for N-linked glycosylation in the F1 subunit polypeptide and none in the F2 subunit polypeptide. The lack of a potential site of glycosylation in F2 makes this protein unique compared to other reported paramyxoviral F proteins. The HN gene extends 1888 nucleotides and encodes a protein consisting of 572 amino acids (mol wt 64,255). This protein contains four potential sites for N-linked glycosylation.

cDNA clones containing the complete coding sequences for the human parainfluenza virus type 3 (PIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes were inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of the P7.5 early-late vaccinia virus promotor. The recombinant vaccinia viruses, designated vaccinia-F and vaccinia-HN, expressed glycoproteins in cell culture that appeared to be authentic with respect to glycosylation, disulfide linkage, electrophoretic mobility, cell surface expression, and, in the case of the HN protein, biological activity. Cotton rats inoculated intradermally with vaccinia-HN developed serum neutralizing antibody titers equal to that induced by respiratory tract infection with PIV3, whereas animals receiving vaccinia-F had threefold lower neutralizing antibody titers. A single immunization with either recombinant vaccinia virus induced nearly complete resistance in the lower respiratory tract of these animals. With regard to protection in the upper respiratory tract, animals immunized with vaccinia-HN or vaccinia-F exhibited reductions in PIV3 replication of greater than 3,000-fold and 6-fold, respectively. This large difference (greater than 500-fold) in reduction of PIV3 replication in the upper respiratory tract was in contrast to the relatively modest difference (3-fold) in serum neutralizing antibody titers induced by vaccinia-HN versus vaccinia-F. This dissociation between the level of neutralizing antibodies and protection suggested that immunity to PIV3 is complex, and that immune mechanisms other than serum neutralizing antibodies make important contributions to resistance to infection. Overall, under these experimental conditions, vaccinia-HN induced a substantially more protective immune response than did vaccinia-F.

The fusion (F) glycoprotein of human parainfluenza type 3 (PI3) virus was produced in insect cells using a baculovirus expression vector (pAcYM1). The recombinant glycoprotein was identified by its reactivity with specific monoclonal and polyclonal antibodies and showed an apparent molecular mass of 70 kDa. Although the fusion protein was found on the infected cell surface, it did not appear to be proteolytically cleaved to F1 and F2 subunits. Immunization of hamsters with the recombinant protein elicited antibody which neutralized infectivity and blocked fusion of virus-infected cells. The protective response to challenge infection of immunized hamsters was similar to that observed with affinity purified F from PI3 virus (Ray et al., J. Virol. 62, 783-787, 1988).

The complete nucleotide sequence of the phosphoprotein (P) mRNA of human parainfluenza virus 3 (PIV-3) was derived from two cDNA clones spanning almost the entire P gene. The mRNA, excluding the poly(A) tail, is 2014 nucleotides long and is bicistronic. The first open reading frame (ORF) codes for the phosphoprotein (P) of mol wt 68,860. Seven nucleotides downstream from the first AUG codon, in a +1 reading frame, there is an additional ORF which can code for a polypeptide of mol wt 23,266. The latter protein appears to be similar to the C proteins found in cells infected with several paramyxoviruses. Comparison of the predicted amino acid sequence of the P and C proteins of PIV-3 with the corresponding Sendai virus proteins reveals considerable homology at the C-terminal half. In contrast, the P and C proteins of PIV-3 share very little homology with the measles virus P and C proteins, respectively.

The sequence of 1690 nucleotides from the 5' end of the viral complementary RNA for the human parainfluenza 3 virus was determined by molecular cloning. One large open reading frame consisting of 1548 nucleotides was demonstrated. The encoded protein, the nucleocapsid protein (NP), consists of 515 amino acids, and has a predicted molecular weight of 57,819. A noncoding 5' sequence of 51 nucleotides is present at the end of the NP-mRNA. Two consensus sequences were identified which are homologous with sequences found in Sendai virus. One of these sequences, AGGATTAAAG, was located at the 5' end of the nucleocapsid mRNA and may function in transcription initiation. The other consensus sequence, GTAAGGGAA, was found in the viral genomic leader sequence. The nucleocapsid protein amino acid sequence was compared to other members of the Paramyxoviridae family. The parainfluenza 3 virus protein nucleocapsid amino acid sequence demonstrated a high degree of homology with the Sendai virus nucleocapsid protein. Seventy percent of the first 387 amino acids from the amino termini were identical. Little homology was observed in the distal carboxy termini.

The sequence of the major nucleocapsid protein (NP) mRNA and its encoded protein were deduced by sequencing a cDNA clone representing the complete mRNA. The cDNA sequence was confirmed by dideoxynucleotide sequencing of purified viral genomic RNA by primer extension using synthetic oligonucleotides. The NP mRNA contains 1,641 nucleotides exclusive of poly(A) and encodes an NP protein of 515 amino acids. Alignment of the human parainfluenza type 3 virus (PF3) NP protein sequence with that of Sendai virus showed that the two proteins shared considerable sequence identity (58.8%). Additional comparisons provided highly significant statistical evidence that the PF3 NP protein sequence is related to those of measles and canine distemper viruses, but there was no evidence of relatedness with the nucleocapsid proteins of respiratory syncytial virus, influenza B virus, or vesicular stomatitis virus.

Antiviral effects of recombinant DNA-derived bovine (Bo) and human (Hu) interferons (IFN) on the replication of bovine herpesvirus-1, parainfluenza-3, and respiratory syncytial viruses were studied. Bovine monolayer cultures were treated with recombinant DNA-produced Bo IFN-alpha 1, Bo IFN-beta 2, Hu IFN-alpha A, or Hu IFN-alpha A/D and then challenge exposed with bovine herpesvirus-1, bovine parainfluenza-3 virus, bovine respiratory syncytial virus, or vesicular stomatitis virus. Treatment with each IFN reduced the viral yield for each of these viruses, compared with that of control cultures.

We investigated properties of the ts mutants that were selected during the course of persistent infection of Vero cells by parainfluenza virus type 3. The mutants demonstrated leakiness when infecting cells at high MOI and interfered with the growth of wild type virus, apparently by inhibiting a step prior to RNA synthesis.

The hemagglutinin-neuraminidase (HN) gene of human type 3 parainfluenza virus has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. HN protein produced in insect cells by the recombinant baculovirus appeared to be glycosylated, was transported to the cell surface, and was biologically active. All of the HN epitopes previously mapped functionally to a region(s) involved in neuraminidase and/or hemagglutination activities were conformationally unaltered on the recombinant protein. The HN produced in this system also induced a protective immune response in immunized cotton rats. From these studies we conclude that the HN expressed in insect cells represents a source of authentic HN glycoprotein suitable for structural analysis and immunization.

The sequence of the matrix (M) protein gene and contiguous intergenic regions of the human parainfluenza 3 virus (PF3) was determined by molecular cloning. The encoded M protein contains 354 amino acids and has a predicted mol wt of 39,506. The M protein amino acid sequence was compared to the homologous proteins from other members of the Paramyxoviridae family. The PF3 protein shared 61% homology with the Sendai virus protein and approximately 35% homology with measles and canine distemper virus proteins. Little homology was observed with respiratory syncytial virus. The M protein appears to be the most highly conserved among the Paramyxoviridae proteins.

The highly syncytium-inducing M strain and the weakly syncytium-inducing SC strain of bovine parainfluenza 3 virus differ by a single amino acid substitution in each of the hemagglutinin-neuraminidase (HN) and membrane (M) proteins, while their fusion (F) proteins are identical (T. Shioda, S. Wakao, S. Suzu, and H. Shibuta, Virology 162:388-396, 1988). We constructed recombinant vaccinia viruses which express separately the M virus HN (Vac-MHN), SC virus HN (Vac-SCHN), M virus M (Vac-MM), SC virus M (Vac-SCM), and common F (Vac-F) proteins. CV-1 cells were infected with the recombinants, singly or in combination, and implanted onto indicator MDBK cells for syncytium formation. Combinations of Vac-MHN plus Vac-F and Vac-SCHN plus Vac-F induced extensive and weak syncytium formation, respectively. Vac-F alone did not induce syncytium formation, and both Vac-MM and Vac-SCM had no effect on syncytium formation. These findings indicated that the syncytium formation by bovine parainfluenza 3 virus requires both the F and HN proteins and that the extensive syncytium formation by the M virus is due to the M virus HN protein. MSC, another weakly syncytium-inducing virus variant, newly isolated from the M virus, was identical to the M virus in the primary structure of the HN and M proteins but differed from the M virus by a single amino acid residue in the F protein. The combination of the recombinant vaccinia virus expressing the MSC virus F protein and Vac-MHN resulted in weak syncytium formation.

The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.

By analysing complementary DNA clones constructed from genomic RNA of bovine parainfluenza 3 virus (BPIV3), we determined the nucleotide sequence of the region containing the entire F and HN genes. Their deduced amino acid sequences showed about 80% homologies with those of human parainfluenza 3 virus (HPIV3), about 45% with those of Sendai virus, and about 20% with those of SV5 and Newcastle disease virus (NDV), indicating, together with the results described in the preceding paper on the NP, P, C and M proteins of BPIV3, that BPIV3, HPIV3 and Sendai virus constitute a paramyxovirus subgroup, and that BPIV3 and HPIV3 are very closely related. The F and HN proteins of all these viruses, including SV5 and NDV, however, were shown to have protein-specific structures as well as short but well-conserved amino acid sequences, suggesting that these structures and sequences are related to the activities of these glycoproteins.

The sequences of the human parainfluenza virus type 3 (PIV3) matrix (M) mRNA [1150 nucleotides exclusive of poly(A)] and predicted M protein (353 amino acids) were determined by sequence analysis of cloned cDNA and viral genomic RNA. The gene-end sequence of the M gene differed from the semi-conserved gene-end sequence of the other PIV3 genes by an apparent insertion of eight nucleotides. The PIV3 M protein shared high sequence homology with Sendai virus and moderate homology with measles virus and canine distemper virus. Statistical analysis of the available sequences showed that the M protein was the most highly conserved parainfluenza viral protein.

The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein-synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease.

cDNA clones of mRNAs for the major nucleocapsid protein (NP), the nucleocapsid P protein plus the nonstructural C protein (P+C), and the matrix protein (M) of human parainfluenza virus type 3 (PF3) were identified by hybrid arrest and hybrid selection of in vitro translation. Previously, cDNA clones were identified and sequenced for the hemagglutinin-neuraminidase glycoprotein (HN) and the fusion glycoprotein (F) mRNAs (N. Elango, J. E. Coligan, R. C. Jambou, and S. Venkatesan, J. Virol. 57:481-489, 1986; M. K. Spriggs, R. A. Olmsted, S. Venkatesan, J. E. Coligan, and P. L. Collins, Virology 152:241-251, 1986). Synthetic oligonucleotides, designed from nucleotide sequences of the cDNAs, were used to direct dideoxynucleotide sequencing of gene junctions in PF3 genomic RNA (vRNA). From sequencing of vRNA, a sixth viral gene was detected and identified as the large nucleocapsid protein (L) gene by hybridization of a synthetic oligonucleotide to intracellular PF3 mRNAs separated by gel electrophoresis. The order of the six PF3 genes on vRNA was 3'-NP-P+C-M-F-HN-L-5'. The five intergenic regions consisted of the trinucleotide 3'-GAA. The PF3 genes initiated with semiconserved 10-nucleotide gene-start sequences and terminated with semiconserved 12-nucleotide gene-end sequences. The M gene terminated with an aberrant gene-end sequence; analysis of intracellular mRNA showed that this aberrant sequence correlated with a disproportionately high accumulation of readthrough mRNA. These studies showed that PF3 encodes six unique mRNAs (NP, P+C, M, F, HN, and L) that encode seven proteins (NP, P, C, M, F, HN, and L) and provided evidence of a close relationship between PF3 and Sendai (murine parainfluenza type 1) viruses.

The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5' terminal sequence of PIV-3 NP mRNA (5'-AGGATTAAAG-3') was similar to the conserved sequence (formula; see text) found at the 5' termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3' terminal tetranucleotide (5'-TAAG-3') preceding the poly (A) tail.

The nucleotide sequence of the gene encoding the fusion (F) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from cDNA clones obtained from virion RNA. The gene is 1792 nucleotides long, including mRNA start and polyadenylation signals typical of paramyxoviruses. The single open reading frame encodes a polypeptide of 553 amino acids, with a predicted molecular weight of 59042. The F polypeptide has three regions of high hydrophobicity: an N-terminal signal peptide, the N terminus of F1 (known from protein sequencing) and a C-terminal membrane-spanning region by which the F glycoprotein is anchored to the membrane. The cleavage site of F0 is located in a highly basic region of the F polypeptide. Five potential asparagine-linked glycosylation sites are present in the amino acid sequence, of which one is in F2 and the others in F1. Comparison of the NDV F amino acid sequence to those from other paramyxoviruses reveals homology to Sendai virus, simian virus 5 and human respiratory syncytial virus. There is also limited homology between the N terminus of F1 of NDV and the N termini of HA2 of influenza viruses. Post-translational modifications of the NDV F polypeptide are discussed in the light of information provided by the amino acid sequence.

The complete sequences of the fusion (F) mRNA and protein of human parainfluenza virus type 3 (PF3) were determined from overlapping cDNA clones. To confirm the cDNA sequence, the complete sequence of the F gene was determined independently by dideoxynucleotide sequencing of genomic RNA using synthetic oligonucleotide primers. The mRNA contains 1845 nucleotides, exclusive of poly (A), has an unusually long (193-nucleotide) 5' nontranslated region, and encodes an F0 protein of 539 amino acids. The site within F0 of the proteolytic cleavage that activates fusion activity was established by direct amino acid sequencing of the NH2 terminus of the F1 subunit. The PF3 F0 protein shares major structural features with the previously sequenced F0 proteins of Sendai virus (murine parainfluenza type 1) and simian virus 5 (SV5, canine parainfluenza type 2), including: similarity in overall length; similarity in location of the site of the activating proteolytic cleavage; the presence of an NH2-terminal signal peptide and COOH-proximal membrane anchor; strong conservation of the sequence at the NH2 terminus of the F1 subunit; and nearly exact conservation in the number and positions of cysteine residues. Alignment of the F0 protein sequences of PF3 with those of Sendai, SV5, and respiratory syncytial virus (RSV) using a matrix that scores both amino acid matches and mismatches provided highly significant statistical evidence that all four proteins are related. The order of decreasing relatedness to PF3 was found to be: Sendai virus, SV5, and RSV.

Cytoplasmic poly(A)-containing RNA from parainfluenza virus 3-infected cells was used as template to construct a cDNA library that was cloned into the EcoRV site of the plasmid pMG5. The resulting clones were screened with [32P]-labeled cDNA probes made from infected and mock-infected cell mRNAs. The virus specificity of the clones was confirmed by Northern blot hybridization. The viral clones were grouped into five different families by hybridization with individual size-selected reverse transcripts representing the major classes of poly(A)+-RNA from virus-infected cells. The five groups were shown to be unrelated on the basis of cross-colony hybridization and corresponded to five unique classes of intracellular viral poly(A)+-RNAs. Clones representing the NP and P genes of PIV-3 were identified by both hybrid-selected and hybrid-arrested translation. Clones specific for the P gene selected mRNA that directed the synthesis of P protein and another polypeptide of 21 kDa. This additional polypeptide comigrated with protein VP8 previously identified in virions and in infected cell lysates.

The sequence of the mRNA encoding the phosphoprotein (P protein) of the human parainfluenza virus 3 (PF3) was determined by molecular cloning. In other Parmyxoviridae the P protein mRNA is functionally bicistronic and encodes an additional smaller nonstructural protein termed C. In this report three open reading frames (ORF) are described. These consist of a single long ORF encoding the P protein, and two shorter ORFs encoding the structural Vp18 protein (analogous to the Sendai C protein) and a putative polypeptide termed D protein. The encoded phosphoprotein consists of 603 amino acids and has a predicted molecular weight of 67,683. The C protein consists of 199 amino acids and has a predicted molecular weight of 23,288. The D protein consists of 140 amino acids and has a predicted molecular weight of 16,270. Although the D protein has not yet been demonstrated in vivo its synthesis could be demonstrated in vitro using a rabbit reticulocyte lysate system. Thus it appears that unlike the other paramyxoviruses, the PF3 P protein mRNA may be functionally tricistronic.

The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) has been determined. The HN gene is 2031 nucleotides long, approximately 13.5% of the viral genome. The nucleotide sequence contains a single long open reading frame which would encode a protein of 577 amino acids, with a mol. wt. of 63,149. This is in good agreement with estimates of the molecular weight of the unglycosylated HN protein. Analysis of the amino acid sequence reveals six potential glycosylation sites and shows the major hydrophobic region to be close to the N terminus. This provides evidence for the N-terminal attachment of HN to the viral membrane. The hydrophilic nature of the extreme N-terminal amino acids suggests the absence of a cleaved signal sequence. Analysis of the long non-coding region at the 3' end of the mRNA encoded by the HN gene of NDV suggests a possible explanation for the origin of HN0 in extremely avirulent strains of NDV. There are regions of high homology between the deduced amino acid sequence of the NDV HN glycoprotein and the HN glycoproteins of two other paramyxoviruses, Sendai virus and simian virus 5 (SV5). An alignment of the HN amino acid sequences of these viruses shows 32% of amino acid residues are conserved between NDV and SV5, and 23% between NDV and Sendai virus. In contrast, only very limited homology is found between NDV HN and the influenza virus glycoproteins.

We have sequenced the coding and noncoding regions of the hemagglutinin-neuraminidase (HN) genes of six clinical strains of human type 3 parainfluenza virus (PIV3) isolated between 1973 and 1983, and compared them to the prototype 1957 strain. Sequence variability does not result from the accumulation of mutations over time, but represents genetic heterogeneity in HN genes within the PIV3 population. Most of the nucleotide diversity occurs in the 5' noncoding sequences, exclusive of regions supplying transcriptional and translational control elements. Although the overall amino acid homology among HN proteins is very high, most variability is concentrated in domains at the carboxyl and amino terminus. This uneven distribution of amino acid diversity may reflect both functional and structural constraints on different HN domains and the epidemiologic features of PIV3 infection.

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.

The nucleotide sequence of the mRNA coding for the fusion glycoprotein (F) of the paramyxovirus, simian virus 5, has been obtained. There is a single large open reading frame on the mRNA that encodes a protein of 529 amino acids with a molecular weight of 56,531. The proteolytic cleavage/activation site of F, to yield F2 and F1, contains five arginine residues. Six potential glycosylation sites were identified in the protein, two on F2 and four on F1. The deduced amino acid sequence indicates that F is extensively hydrophobic over the length of the polypeptide chain. Three regions are very hydrophobic and could interact directly with membranes: these are the NH2-terminal putative signal peptide, the COOH-terminal putative membrane anchorage domain, and the NH2-terminal region of F1.

The sequence of the gene encoding the L protein of the human parainfluenza 3 virus was determined by direct dideoxy sequence analysis of the genomic 50 S RNA and confirmed by molecular cloning and sequence analysis of recombinant clones. A series of three overlapping clones was generated by primer extension using genomic 50 S RNA as the template. These clones originate within the 5' end of the hemagglutinin-neuraminidase gene, span the entire L gene, and extend into the extracistronic 5' end of the viral RNA. The L gene extends 6755 nucleotides (inclusive of the putative transcription initiation and polyadenylation signal sequences) and encodes a protein consisting of 2233 amino acids (MW 255,812). There are 44 nucleotides downstream of the putative polyadenylation signal sequence which may represent a negative-strand leader. The complementary sequence of the extracistronic region is nearly identical to the 3' end of the viral RNA. Thirty-three of the first thirty-nine nucleotides of the 3' ends of the plus and minus strands are conserved. Comparison of amino acid sequence homology with other paramyxoviral L proteins indicates a high degree of sequence conservation with Sendai virus (62%) and Newcastle disease virus (28%). In addition, four smaller regions were identified which shared extensive homology with the L protein of vesicular stomatitis virus, a member of the Rhabdoviridae family.

The nucleotide sequence of the human parainfluenza virus 3 (HPIV3) hemagglutinin-neuraminidase (HN) gene has been determined using cDNA clones derived from both HPIV3 genomic RNA and mRNA. The HN mRNA contains 1,882 nucleotides, not including the poly(A) tail. Primer extension experiments were carried out to locate the 5' terminal nucleotide of the HN mRNA. The 3' end of the mRNA was located at a putative polyadenylation signal. The HPIV3 HN mRNA has one large open reading frame that codes for 572 amino acids with a deduced molecular weight of 64,178. Potential polymerase recognition signals for the HN and L genes were located in the flanking regions. The HN protein of HPIV3 shares some common features with the previously sequenced HN proteins of Sendai virus and Simian virus 5. The features include: an N-terminal membrane anchor, two regions of highly conserved amino acid sequence and strong conservation in the positions of the cysteine residues. The relationship is closest between Sendai virus and HPIV3.

The complete nucleotide sequence of the human parainfluenza virus type 3 (HPIV3) fusion (F) protein gene has been determined. The HPIV3 F gene is 1851 nucleotides long including six U residues in the genomic RNA, which probably direct synthesis of the first few nucleotides in the F mRNA polyadenylate tail. The HPIV3 F gene contains a single long open reading frame coding for 539 amino acids. The predicted molecular weight of the unglycosylated precursor F0 protein was 60031. Four potential carbohydrate acceptor sites were identified. Comparison of the HPIV3 F protein sequence with the F gene sequences of two other paramyxoviruses, Sendai virus and simian virus 5, indicated a very close evolutionary relationship between HPIV3 and Sendai virus. Sequence analysis of HPIV3 F gene flanking regions identified signals which appear to be responsible for polymerase recognition and polyadenylation.

We have previously identified 11 epitopes located in two topologically nonoverlapping antigenic sites (A and B) and a third bridging site (C) on the human type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) glycoprotein by using monoclonal antibodies (MAbs) which inhibit hemagglutination and virus infectivity (K. L. Coelingh, C. C. Winter, and B. R. Murphy, Virology 143:569-582, 1985). We have identified three additional antigenic sites (D, E, and F) on the HN molecule by competitive-binding assays of anti-HN MAbs which have no known biological activity. Epitopes in sites A, D, and F are conserved on the bovine PIV3 HN glycoprotein and also among a wide range of human isolates. The dideoxy method was used to identify nucleotide substitutions in the HN genes of antigenic variants selected with neutralizing MAbs representing epitopes in site A which are shared by human and bovine PIV3. The deduced amino acid substitutions in the variants were located in separate hydrophilic stretches of HN residues which are conserved in the primary structures of the HN proteins of both human and bovine PIV3 strains.

Vero cells persistently infected with parainfluenza virus type 3 (para 3) were examined for the production of temperature-sensitive (ts) mutants. After 6 months and 33 passages, ts mutants formed the great majority of the virus being shed into the supernatant fluid. Complementation studies gave evidence that the mutants shared a common lesion. Although ts mutants may play a role in the maintenance of persistent infection in this system, the mutants proved to be unstable when removed from the milieu of persistent infection. It is thus possible that their selection may be secondary to (an) unidentified factor(s) which play(s) a more primary role in the maintenance process.

The complete sequence of the gene encoding the matrix protein (M) of human parainfluenza virus type 3 (PIV-3) was determined from cDNA clones and from primer extension dideoxy sequencing of the viral genome. The M mRNA is 1150 nucleotides in length, exclusive of polyadenylate, and codes for a protein of 353 amino acids, having a calculated molecular weight of 39,480. The M protein of PIV-3 was found to have a high degree of sequence homology with that of a closely related paramyxovirus, Sendai virus, and to a lesser extent it contained sequence homology with two more distant paramyxoviruses, measles virus and canine distemper virus. We also determined the sequences of the intergenic junctions for the first four genes of PIV-3: NP, P, M, and F. Comparison of these sequences yielded a consensus mRNA start sequence of 5'-AGGANNAAAGA-3', an mRNA end sequence of 5'-UAAGAAAAA-3', and an intergenic sequence of 5'-CUU-3'. The end sequence of the M gene is unusual in that it contains an eight base insertion prior to the A5 tract found in the consensus sequence. This disruption appears to cause a high frequency of readthrough by the viral transcriptase at this junction.

We present the nucleotide sequence of bovine parainfluenza 3 virus (BPIV3) genome from its 3' end to the opening region of the F gene, through the NP, P plus C, and M genes. Comparison of the sequence with those reported for other paramyxoviruses indicated that BPIV3 was most similar to human parainfluenza 3 virus (HPIV3), and also very similar to Sendai virus in the structural make-up of its genome and the amino acid sequences of its gene products, suggesting that these three viruses constitute a paramyxovirus subgroup from which Newcastle disease and measles viruses are separable. In BPIV3 and Sendai virus, the NP and M proteins, the main structural elements, were more highly conserved than the functionally important P and C proteins. This tendency was also observed even in BPIV3 and HPIV3. Virus-specific amino acid sequences of the NP and M proteins were found at the carboxyl and amino terminal regions, respectively. BPIV3 M mRNA was found to have aberrations in its poly A attachment site.