Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.     

Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein

The coronavirus infectious bronchitis virus (IBV) nucleocapsid (N) protein is an RNA binding protein which is phosphorylated at two conserved clusters. Kinetic analysis of RNA binding indicated that the C-terminal phosphorylation cluster was involved in the recognition of viral RNA from non-viral RNA. The IBV N protein has been found to be essential for the successful recovery of IBV using reverse genetics systems. Rescue experiments indicated that phosphorylated N protein recovered infectious IBV more efficiently when compared to modified N proteins either partially or non-phosphorylated. Our data indicate that the phosphorylated form of the IBV N protein plays a role in virus biology.     

Association of the infectious bronchitis virus 3c protein with the virion envelope

A highly purified radiolabeled preparation of the coronavirus infectious bronchitis virus (IBV) was analyzed, by immunoprecipitation with monospecific antisera, for the presence of a series of small virus proteins recently identified as the products of IBV mRNAs 3 and 5. One of these, 3c, a 12.4K protein encoded by the third open reading frame of the tricistronic mRNA3 was clearly detectable and was found to cofractionate with virion envelope proteins on detergent disruption of virus particles. These results, together with the hydrophobic nature of 3c and its previously demonstrated association with the membranes of infected cells, suggest strongly that 3c represents a new virion envelope protein, which may have counterparts in other coronaviruses.     

Functional domains in the feline immunodeficiency virus nucleocapsid protein

Retroviral nucleocapsid (NC) proteins are small Gag-derived products containing one or two zinc finger motifs that mediate genomic RNA packaging into virions. In this study, we addressed the role of the feline immunodeficiency virus (FIV) NC protein in the late stages of virus replication by analyzing the assembly phenotype of FIV NC mutant viruses and the RNA binding activity of a panel of recombinant FIV NC mutant proteins. Substitution of serine for the first cysteine residue in the NC proximal zinc finger was sufficient to impair both virion assembly and genomic RNA binding. A similar defective phenotype with respect to particle formation and RNA binding was observed when the basic residues Lys28 and Lys29 in the region connecting both zinc fingers were replaced by alanine. In contrast, mutation of the first cysteine residue in the distal zinc finger had no effect on virion production and allowed substantial RNA binding activity of the mutant NC protein. Moreover, this NC mutant virus exhibited wild-type replication kinetics in the feline MYA-1 T-cell line. Interestingly, amino acid substitutions disrupting the highly conserved PSAP and LLDL motifs present in the C-terminus of the FIV NC abrogated virion formation without affecting the NC RNA binding activity. Our results indicate that the proximal zinc finger of the FIV NC is more important for virion production and genomic RNA binding than the distal motif. In addition, this study suggests that assembly domains in the FIV NC C-terminus may be functionally equivalent to those present in the p6 domain of the Gag polyprotein of primate lentiviruses.     

Antigenicity of the peplomer protein of infectious bronchitis virus

To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited DNase digestion and inserted in the Escherichia coli expression plasmid pEX (Stanley and Luzio, 1984). The antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. The polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. The N-terminal region of one of the two subunits, S2, was recognized by all polyclonal sera and by two monoclonal antibodies. This clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. The epitopes found as antigenic pEX expression products do not coincide with the regions in the S1 subunit that have been found to contain hypervariable sequences. We suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pEX system. The relevance of our findings for vaccine development is discussed.     

Enhancement of the immunogenicity of an infectious bronchitis virus DNA vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin-2

A DNA vaccine against infectious bronchitis virus (IBV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To develop a more potent IBV DNA vaccine formulations, a monocistronic vector encoding the nucleocapsid protein of IBV and a bicistronic vector separately encoding the nucleocapsid protein and immune-stimulatory interleukin-2 were constructed. When the DNA vaccines were administered to the quadriceps muscle of chickens, the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels elicited by either monocistronic or bicistronic DNA vaccines. The percentage of CD3+, CD3+CD8+ and CD3+CD4+ subgroups of peripheral blood T-lymphocytes in chickens immunized with bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent strain of IBV, the protective efficacy could be enhanced significantly after immunization with bicistronic DNA vaccine. These results demonstrated that bicistronic DNA vaccine is an effective approach to increase IBV DNA vaccine immunogenicity.     

Comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses.

The natural sequence variations of the nucleocapsid genes of the Gray, Arkansas99 (Ark99), and Holland52 (Holl52) strains of infectious bronchitis virus (IBV) were determined. These were compared with previously published sequencing data of other IBV strains, as well as other coronaviruses, in order to correlate the serological and evolutionary relationship of coronaviruses. IBV nucleotide sequence alignment shows that overall the sequences are highly conserved, with homologies from 91.1 to 96.5%. However, there are also two regions (730 to 800 and 1138 to 1166) that appear to be even more highly conserved. Overall, the nucleocapsid protein is highly variable both in size and composition between coronavirus major antigenic groups but is conserved within these groups. A phylogenetic tree of the nucleocapsid protein of various coronaviruses indicates that the coronaviruses fall into distinct groups that correspond to the three major antigenic groups; however, a phylogenetic tree of the IBV nucleocapsid shows that this does not hold true for the type specific antigenic groups of IBV.     

Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus

Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75-43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.     

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.     

Moloney murine leukemia virus genomic RNA packaged in the absence of a full complement of wild type nucleocapsid protein

The current model for MLV genomic RNA (gRNA) packaging predicts that of the thousands of Gag proteins in a budding virion, only a small number (≤1%) may be necessary to recruit gRNA. Here, we examined the threshold limits of functional Gag required to package gRNA using wild-type (WT) and packaging deficient mutant nucleocapsid (NC) phenotypically mixed virions. Although gRNA packaging was severely diminished for the NC mutant, the residual encapsidated RNA dimer displayed motility on gels, thermostability, and integrity that was indistinguishable from that of WT. In phenotypically mixed virions, gRNA encapsidation recovered to within approximately two-fold of WT levels when the amount of WT NC was 5-10% of the total. Our results demonstrate that NC's roles in gRNA dimerization and packaging are genetically separable. Additionally, MLV gRNA packaging does not require 100% WT NC, but the amount of functional NC required is greater than the predicted minimum.     

The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs

Cis-acting elements that regulate translation have been identified in the 3' noncoding regions (NCRs) of cellular and viral mRNAs. As one means of analyzing the effect on translation of the conserved 3' terminal RNA structure of the West Nile virus (WNV) genome, the translation efficiencies of chimeric mRNAs composed of a CAT reporter gene flanked by viral or nonviral 5' and 3' terminal sequences were compared. In vitro, the WNV 3'(+) stem loop (SL) RNA reduced the translation efficiencies of chimeric mRNAs with either viral or nonviral 5' NCRs, suggesting that a specific 3'-5' RNA-RNA interaction was not involved. In contrast, the 3' terminal sequence of a togavirus, rubella virus, enhanced translation efficiency. The WNV 3'(+)SL reduced translation efficiency both in cis and in trans and of both capped and uncapped chimeric mRNAs. We have previously reported that three cellular proteins bind specifically to the WNV 3'(+)SL RNA. Competition between the WNV 3'(+)SL and the 5' terminus of the chimeric mRNAs for proteins involved in translation initiation could explain the translation inhibition observed.     

Detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay

The recombinant antigen obtained by cloning and expressing two IBV nucleocapsid protein fragments (143-414 aa, 281-414 aa) in Escherichia coli was used for the detection of avian infectious bronchitis virus (IBV) specific antibodies in chicken sera by the indirect ELISA (rNpIBV-ELISA). As a result of testing 1524 serum samples the diagnostic sensitivity and specificity of rNpIBV-ELISA when comparing those of the routine whole IBV ELISA have been shown to be 93.81% and 87.36%, respectively. The agreement value was 91.5%.