Sequence characterization of the membrane protein gene of paramyxovirus simian virus 5

The complete nucleotide sequence of the membrane (M) protein gene of the paramyxovirus simian virus 5 (SV5) was determined from cDNA clones of viral mRNAs. The M gene boundaries were determined by (i) primer extension sequencing on M mRNA; (ii) nuclease S1 analysis; and (iii) primer extension sequencing on viral genomic RNA. The M gene mRNA consisted of 1371 templated nucleotides. It contains a single large open reading frame that can encode a protein of 377 amino acids with a predicted Mr = 42,253. The authenticity of the predicted M protein coding sequence was confirmed by synthesis of the M protein from mRNA synthesized from cDNA. The predicted M amino acid sequence indicated it is an overall hydrophobic protein carrying a net positive charge. Alignment of the SV5 protein amino acid sequence with the M protein sequences of other paramyxoviruses indicated that these viruses fall into the following two groups: (1) SV5, mumps virus, and Newcastle disease virus; or (2) Sendai, parainfluenza virus type 3, measles virus, and canine distemper virus, with mumps virus M sequence being the most closely related to SV5.     

Analysis and gene assignment of mRNAs of a paramyxovirus, simian virus 5

Polypeptides synthesized by the paramyxovirus SV5 in infected CV-1 cells were readily identified when the host cell was treated with actinomycin D. The unglycosylated forms of HN and Fo synthesized in infected cells in the presence of tunicamycin and HN and Fo synthesized in vitro were identified by immunoprecipitation with specific antibodies. Separation of SV5-specific poly(A)-containing RNAs on methyl-mercury agarose gels and in vitro translation of fractions, indicated that the viral polypeptides were translated from individual mRNAs except P (Mr approximately 44K) and the nonstructural polypeptide V (Mr approximately 24K) for which the mRNAs could not be separated. cDNA copies of SV5-specific mRNAs were synthesized and cloned in plasmid pBR322. Clones to NP, P + V, M, F, and HN were identified by hybrid-arrest and hybrid-selection translation of SV5 mRNAs. Tryptic peptide mapping of polypeptides P and V indicated that the peptides of V were a subset of those of P. Hybridization of cDNA probes to infected cell mRNAs separated on agarose gels permitted identification of the NP, P + V, M, F, and HN mRNAs and presumptive polycistronic mRNAs. The sizes and sequence homologies of these polycistronic mRNAs were used to derive a likely gene order on the SV5 50 S genome RNA.     

Mapping of domains on the human parainfluenza type 2 virus P and NP proteins that are involved in the interaction with the L protein

Eleven monoclonal antibodies (MAbs) directed against the large (L) protein of human parainfluenza type 2 virus (hPIV-2) were prepared to examine the interactions of the L protein with other viral proteins. Coimmunoprecipitation assays using these MAbs revealed that the L protein directly interacted with the phospho- (P) and nucleocapsid (NP) proteins in vivo and in vitro. Mutational analysis of the P or NP protein was performed to identify the region(s) on these proteins interacting with L protein, indicating that amino acids 278-353 on the P protein and amino acids 403-494 on the NP protein are essential for the binding to the L protein.     

Distinct pathways for signaling maturation in macrophages and dendritic cells after infection with paramyxovirus simian virus 5

Professional antigen-presenting cells are critical components of both the innate and adaptive immune responses. Although dendritic cells (DCs) are generally thought to be the primary activators of naive T cells, macrophages have also been shown to fulfill this role. As with DCs, the capacity to induce optimal activation of T cells requires that macrophages undergo a process that results in the increased expression of costimulatory molecules, such as CD40, CD80, and CD86, and the production of cytokines. In this study we analyzed the effect of infection of macrophages generated from BALB/c mice with the paramyxovirus simian virus 5 (SV5). Here we have shown that bone marrow-derived macrophages (BMMs) are not productively infected at any multiplicity of infection tested. Analysis of activation markers revealed that SV5-infected BMMs robustly upregulated CD40 and modestly upregulated CD86, but did not upregulate the expression of CD80. Further, SV5-infected BMMs secreted low levels of interferon-beta and interleukin (IL)-12p40, but high levels of tumor necrosis factor-alpha and IL-6. Intriguingly, upregulation of these molecules on BMMs, unlike our previous results using bone marrow-derived dendritic cells, was not dependent on live virus. These findings provide evidence that different professional antigen-presenting cells can detect and respond to virus via distinct mechanisms.     

The haemagglutinin-neuraminidase glycoprotein of the porcine paramyxovirus LPMV: comparison with other paramyxoviruses revealed the closest relationship to simian virus 5 and mumps virus

Summary The complete nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of the porcine paramyxovirus LPMV, was determined from cDNA derived from viral genomic RNA. The gene was 1906 nucleotides long including a putative gene end and poly A signal. One long open reading frame was found encoding a protein of 576 amino acids with a calculated molecular weight of 63,324. The protein contains four potential N-glycosylation sites and a major hydrophobic region near the N-terminal, suggesting a membrane anchor domain. Comparison of the deduced amino acid sequence of the LPMV HN protein with that of other paramyxovirus HN proteins, revealed the highest amino acid identity to simian virus 5 of 43% and mumps virus of 41%.     

Sequence homology between polyoma virus, simian virus 40, and a papilloma-producing virus from a Syrian hamster: evidences for highly conserved sequences

Sequence homology between the genomes of a hamster papovavirus (HaPV), polyoma virus (Py), and Simian virus 40 (SV40) has been studied by filter hybridization and electron microscopy under conditions of varying stringency. Hybrids between the HaPV and SV40 DNAs could be demonstrated only under nonstringent conditions. The region of highest homology was mapped in the early region of the SV40 genome. Extensive homology was detected between the genomes of HaPV and Py under stringent hybridization conditions, indicating at least 80% base matching in the regions of strongest sequence homology. These sequences were localized within both the early region and the late region of the Py genome. The homologous DNA segments mapped in the Py and the SV40 genomes are among the most strongly conserved regions in the polyoma (miopapova)-virus group.     

Positive- and negative-acting signals combine to determine differential RNA replication from the paramyxovirus simian virus 5 genomic and antigenomic promoters

The cis-acting signals found at the 3' ends of the genomic and antigenomic RNAs are a major factor determining the level of paramyxovirus RNA replication from each promoter. Using a minigenome system that reconstitutes SV5 RNA synthesis from cDNA-derived components, we show here that the genomic promoter (GP) for the paramyxovirus SV5 directs RNA replication approximately 14-fold lower than that seen from the antigenomic promoter (AGP). The goal of this study was to identify cis-acting signals responsible for differential levels of RNA replication from the SV5 GP and AGP. We have previously shown that the SV5 AGP contains three sequence-dependent elements (CRI, CRII, and Region III) that are separated by sequence-independent spacer regions. Minigenomes containing chimeric promoters were constructed to test the hypothesis that transfer of discrete cis-acting AGP elements to the GP could confer higher replication properties to the GP. Minigenomes containing a substitution of the AGP CRI, CRII, or Region III elements alone in place of the corresponding GP sequences did not show enhanced levels of RNA replication. However, transfer of both the AGP 3' terminal CRI and Region III elements into the corresponding sites of the GP led to a minigenome which replicated to approximately 40% of the levels seen with the AGP. This enhanced RNA replication from the GP was further increased up to AGP levels by also including the intervening AGP segment (bases 20-50) located between CRI and Region III. Importantly, transfer of nonviral sequences in place of GP bases 20-50 also increased RNA replication to levels approaching that of the AGP, but only in the context of the AGP CRI and Region III substitutions. These data indicate that differential levels of RNA replication from the SV5 GP and AGP are due to a combination of positive-acting signals in the AGP (CRI and Region III) and a negative-acting signal in the GP (bases 20-50). Possible functions for the SV5 promoter elements in determining RNA replication levels are proposed.     

A peptide comparison of proteins in the simian virus 40 virion.

Evidence is presented which suggests that (i) simian virus 40 (SV40) virion proteins VP-1 and VP-3 are independently coded; (ii) there is little sequence homology between the nonhistone virion proteins of SV40 and those of polyoma virions; and (iii) two of the histone-like protein species of SV40 are also present in polyoma virions.     

Molecular cloning and sequencing of influenza virus A/Victoria/3/75 polymerase genes: sequence evolution and prediction of possible functional domains

The influenza virus A/Victoria/3/75 (H3N2) polymerase genes encoding PB1, PB2 and PA have been cloned by cDNA synthesis and insertion into bacterial vectors. The complete sequence for each polymerase gene has been obtained from random M13 subclones and compared to other influenza virus polymerase genes. A total of 45, 74 and 78 nucleotide changes were fixed in the period 1968-1975, corresponding to 10, 12 and 9 amino acid changes, for PB1, PB2 and PA genes, respectively. The amino acid sequence of PB1 polypeptide contains motifs found in a series of positive- and negative-RNA virus polymerase genes and that of PA polypeptide share invariant residues common to DNA and presumptive RNA helicases.     

Molecular cloning and sequence analysis of the duck enteritis virus UL5 gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, the DEV UL5 gene was cloned and sequenced from a vaccine virus. According to the consensus sequence of herpesvirus UL5 and UL3 gene degenerate oligonucleotide primers were designed and were used in the polymerase chain reaction (PCR) to amplify DNA products with 4577 bp in size. DNA sequence analysis revealed a 2568 bp open reading frame (ORF) encoding a 855 amino acid polypeptide homologous to herpesvirus UL5 proteins. The DEV UL5 gene has a base composition of 769 adenine (29.95%), 556 cytosine (21.65%), 533 guanine (20.76%) and 710 thymine (27.65%). Sequence comparison revealed that the nucleotide sequence of the DEV UL5 gene was highly similar to other alphaherpesviruses. Phylogenetic tree analysis showed that the fifteen herpesviruses viruses analyzed fell into four large groups, and the duck enteritis virus itself branched and was most closely related to meleagrid herpesvirus 1, gallid herpesvirus 2 and gallid herpesvirus 3 subtrees.     

Antigenic analysis of African measles virus field isolates: identification and localisation of one conserved and two variable epitope sites on the NP protein

Measles virus isolates from epidemics in the Cameroons (1983) and Gabon (1984) were analysed by a panel of monoclonal antibodies against four of the virion proteins. We observed no antigenic variation in the haemagglutinin, the fusion glycoprotein, or in the matrix protein. However, both inter- and intra-epidemic variation was observed in the nucleoprotein (NP). On the basis of strain reactivity and a competition binding assay, three epitopic sites were designated on the NP. One site was found on all the measles virus strains examined, whereas the other two were variable. Examination of proteolytic cleavage of the NP in situ (on the ribonucleoparticle) showed that the conserved site is located on a large fragment which remains bound to the viral genome. The peptide removed by proteolysis contained the two variable epitopes. The variability of the NP is discussed in relationship to its biological activity.     

Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence.

We have cloned and determined the nucleotide sequences of the seventh gene of the Miyahara strain of mumps virus (MuV) encoding the L protein. The L gene is 6925 nucleotides in length and contains a single long open reading frame which is capable of coding for a protein of 2261 amino acids with a calculated molecular weight of 256,571 Da. The deduced amino acid sequence of the L protein of MuV showed significant homology with those of six other paramyxoviruses, human parainfluenza type 2 virus, Newcastle disease virus, Sendai virus, measles virus, human parainfluenza type 3 virus, and human respiratory syncytial virus. The predicted MuV L protein contained distinct elements thought to be essential for RNA polymerase activity. A noncoding sequence of 24 nucleotides downstream of the presumed polyadenylation site of the L gene showed significant complementarity with the leader sequence composed of 55 nucleotide at the 3' end of the genomic RNA.