Functional differences in the peplomer glycoproteins of feline coronavirus isolates.

The feline coronaviruses can be divided into two distinct antigenic groups on the basis of antigenic differences found on the peplomer (E2) glycoprotein of the virus. Because the E2 glycoprotein is responsible for many of the biological functions of coronaviruses, experiments were done to determine whether there were any E2 functional differences between these two antigenic groups. The avirulent feline infectious peritonitis virus (FIPV) isolate FIPV-UCD-2, which has one antigenic type of E2, was less rapidly internalized and could not spread from cell to cell in the presence of neutralizing antibody. Two virulent isolates, FIPV-DF2 and FIPV-79-1146, as well as the non-FIP-causing feline enteric coronavirus (FECV) isolate FECV-79-1683, all of which have the second antigenic type of E2, were very rapidly internalized and were able to spread from cell to cell despite the presence of neutralizing antibody. The avirulent FIPV-UCD-2 and FECV-79-1683 isolates were more labile at 37 degrees C at pHs of 6.5 and above than were the virulent FIPV-DF2 and FIPV-79-1146 isolates.     

Biosynthesis of glycoproteins E and I of feline herpesvirus: gE-gI interaction is required for intracellular transport.

The biosynthesis of glycoproteins E and I of feline herpesvirus was studied by using the vaccinia virus vTF7-3 expression system. gE and gI were synthesized as N-glycosylated, endoglycosidase H (EndoH)-sensitive precursors with Mrs of 83,000 and 67,000, respectively. When coexpressed, gE and gI formed sodium dodecyl sulfate-sensitive hetero-oligomeric complexes that were readily transported from the endoplasmic reticulum (ER). Concomitantly, the glycoproteins acquired extensive posttranslational modifications, including O glycosylation, leading to an increase in their apparent molecular weights to 95,000 and 80,000 to 100,000 for gE and gI, respectively. In the absence of gE, most gI remained EndoH sensitive. Only a minor population became EndoH resistant, but these molecules were processed aberrantly as indicated by their Mrs (100,000 to 120,000). By immunofluorescence microscopy, gI was detected primarily in the ER but also at the plasma membrane. gE, when expressed by itself, remained EndoH sensitive and was found only in the ER and the nuclear envelope. These results were corroborated by studying the biosynthesis of gE in feline herpesvirus (FHV)-infected cells. In cells infected with wild-type FHV, gE acquired the same co- and posttranslational modifications as during vTF7-3-driven expression. However, an FHV mutant lacking gI failed to produce mature gE. We conclude that gE is retained in the ER, presumably by associating with molecular chaperones, and becomes transport competent only when in a complex with gI.     

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.

The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus at 37 degrees C resulted in formation of a complex between one of the viral glycoproteins, E1, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in E1, resulting in aggregation of E1 and interaction with the viral RNA in the nucleocapsid. E1 also bound rRNA. The E1-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein E1 from the peplomeric glycoprotein E2 permitted preparation of antisera against these isolated proteins. A model is proposed for the arrangement of the three major structural proteins in the coronavirus A59 virion in relation to the viral envelope and RNA.     

Disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins.

We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum (ER). Intrachain disulfide bonds are formed in the S protein but not in M, which was demonstrated in a pulse-chase experiment by analyzing the viral proteins under nonreducing conditions. To reduce disulfide bonds in vivo, we added dithiothreitol (DTT) to the culture medium of mouse hepatitis coronavirus-infected cells following a procedure recently described by Braakman et al. (I. Braakman, J. Helenius, and A. Helenius, EMBO J. 11:1717-1722, 1992). Short exposure to DTT resulted in the complete reduction of newly synthesized S protein and affected its conformation as judged by the change in mobility in nonreducing gels and by the loss of recognition by a conformation-specific monoclonal antibody. Using this antibody in an immunofluorescence assay, we monitored the reducing effect of DTT in situ. DTT was found to initially affect only the S protein present in the ER; also, after longer treatment, the remaining signal also gradually disappeared. In contrast, folding and transport of the M protein were not inhibited by DTT. Under reducing conditions, M was transported efficiently to the trans side of the Golgi complex, indicating that cellular processes such as ER-to-Golgi transport, O-glycosylation, and Golgi retention were unaffected. In the presence of DTT, the M protein even moved at an increased rate to the Golgi complex, which is probably because of its failure to interact with unfolded S protein. The effects of in vivo reduction were reversible. When DTT was removed from pulse-labeled cells, the S protein folded posttranslationally and aberrantly; during its oxidation, most of S now transiently aggregated into large disulfide-linked complexes from which subsequently folded S molecules dissociated.     

Reasoning of spike glycoproteins being more vulnerable to mutations among 158 coronavirus proteins from different species

In this study, we used the probabilistic models developed by us over the last several years to analyze 158 proteins from coronaviruses in order to determine which protein is more vulnerable to mutations. The results provide three lines of evidence suggesting that the spike glycoprotein is different from the other coronavirus proteins: (1) the spike glycoprotein is more sensitive to mutations, this is the current state of the spike glycoprotein, (2) the spike glycoprotein has undergone more mutations in the past, this is the history of spike glycoprotein, and (3) the spike glycoprotein has a bigger potential towards future mutations, this is the future of spike glycoprotein. Furthermore, this study gives a clue on the species susceptibility regarding different proteins.Figure Predictable and unpredictable portions in coronavirus proteins. The data are presented as median with interquartile range. * the predictable and unpredictable portions in spike glycoprotein group are statistically different from any other protein groups at p<0.05 level, except for hemagglutinin-esterase precursor group. # the predictable and unpredictable portions in spike glycoprotein group are statistically different from hemagglutinin-esterase precursor, membrane protein and nucleocapsid protein groups at p<0.05 level. the predictable and unpredictable portions in spike glycoprotein group are statistically different from hemagglutinin-esterase precursor, and membrane protein groups at p<0.05 level.Electronic Supplementary Material is available for this article if you access the article at .     

Three independent isolates of feline sarcoma virus code for three distinct gag-x polyproteins.

Cells nonproductively transformed by the Snyder-Theilen, Gardner-Arnstein, and McDonough strains of feline sarcoma virus synthesize gag-x polyproteins of 78,000, 100,000, and 180,000 daltons, respectively. These feline sarcoma virus-coded products were precipitated by antisera to polypeptides encoded by the gag gene of feline leukemia virus and by rat antisera raised to feline sarcoma virus-transformed rat tumor cells. Precipitation with rat antisera absorbed with feline leukemia virus showed that the x-portions of the three gag-x proteins were each antigenically distinct, suggesting that the src genes of the three independent isolates are not identical. Anti-x sera did not precipitate products from radiolabeled cat lymphoid tumor cells (FL74) and therefore lacked reactivity to the feline leukemia virus-induced tumor-specific antigen, FOCMA.     

Antigenic characterization of type C RNA virus isolates of gibbon apes.

Type C RNA viruses initially isolated from a lymphosarcoma of a gibbon ape and from a fibrosarcoma of a woolly monkey are very closely related immunologically. However, recent studies have shown that these viruses are distinguishable in a radioimmunoassay for the 12,000-molecular-weight polypeptide (p12) of the woolly monkey virus. In the present report, an immunoassay has been developed for the p12 polypeptide of the gibbon ape type C virus. This assay is shown to further distinguish the woolly monkey and gibbon ape viruses. In type-specific assays for the p12 polypeptides of these viruses, two new type C viruses isolated from gibbons in a second colony, characterized by high incidence of hemopoietic neoplasia, are immunologically distinguishable from the original gibbon ape virus. The p12 type-specific immunoassays described in the present report may be of importance in studying the natural history of these viruses and their relationship to tumors of primates.     

Inter-strain homology of pilin gene sequences in Neisseria meningitidis isolates that express markedly different antigenic pilus types

Neisseria meningitidis isolates examined in this study elaborated one of two pilus types that were antigenically markedly different. Each pilus type reacted either with SM1, a monoclonal antibody that recognizes an epitope common to all gonococcal pili, or with a polyclonal antiserum raised against meningococcal pili that did not react with SM1, but not both. Total genomic DNA from all N. meningitidis isolates analysed, irrespective of pilus type, contained at least one region with extensive homology to a gonococcal pilE probe. Different N. meningitidis strains possessed one of several configurations of genomic pilE-homologous segments. Chromosomal rearrangement of pilE-homologous sequences was associated with P+ to P- pilus phase transition in the strains examined. The arrangement of pilE-homologous segments in total genomic DNA from N. meningitidis isolated from the blood and cerebro-spinal fluid of the same patient was apparently identical.     

Antigenic analysis of human cytomegalovirus isolates from Japanese women and infants.

The antigenic differences among human cytomegalovirus (CMV), two laboratory strains (Davis, AD 169), isolates from pregnant women's cervical secretions, mother's milk, infants' throat swabs and urine were analyzed by means of the plaque reduction assay using human sera which were assumed to contain monotypic antibodies by primary infection and boosted antibodies by reinfection or reactivation of the latent virus. In the cross-neutralization tests, there were no remarkable differences among the CMV strains examined. Moreover, in the neutralization kinetics, normalized kappa values among the strains constantly exceeded 80. Therefore, it is suggested that the human CMV strains examined in the study were serologically identical or very closely related.     

Differences in mechanisms of transformation by independent feline sarcoma virus isolates.

The Gardner and Snyder-Theilen isolates of feline sarcoma virus (FeSV) have previously been shown to encode high-molecular-weight polyproteins with a transforming function and an associated tyrosine-specific protein kinase activity. Cells transformed by these viruses exhibited morphological alterations, elevated levels of phosphotyrosine, and a reduced capacity for binding epidermal growth factor. In addition, polyproteins encoded by both of these FeSV isolates bound to, and phosphorylated tyrosine acceptor sites within, a 150,000-molecular-weight cellular substrate (P150). McDonough FeSV-transformed cells resembled Gardner and Snyder-Theilen FeSV transformants with respect to morphological changes and a reduced capacity for epidermal growth factor binding. in contrast to the other two FeSV isolates, however, McDonough FeSV encoded as its major translational product a high-molecular-weight polyprotein with probable transforming function but without protein kinase activity detectable under similar assay conditions. Moreover, total cellular levels of phosphotyrosine remained unaltered in McDonough FeSV-transformed cells, and the major McDonough FeSV polyprotein translational product lacked binding affinity for P150. These findings argue for differences in the mechanisms of transformation by these independently derived FeSV isolates.     

Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity.

The degree of antigenic relatedness between human respiratory syncytial virus (RSV) subgroups A and B was estimated from antibody responses induced in cotton rats by respiratory tract infection with RSV. Glycoprotein-specific enzyme-linked immunosorbent assays of antibody responses induced by RSV infection demonstrated that the F glycoproteins of subgroups A and B were antigenically closely related (relatedness, R approximately 50%), whereas the G glycoproteins were only distantly related (R approximately 5%). Intermediate levels of antigenic relatedness (R approximately 25%) were seen in neutralizing antibodies from cotton rats infected with RSV of the two subgroups. Immunity against the F glycoprotein of subgroup A, induced by vaccinia-A2-F, conferred a high level of protection which was of comparable magnitude against challenge by RSV of either subgroup. In comparison, immunity against the G glycoprotein of subgroup A, induced by vaccinia-A2-G, conferred less complete, but significant, protection. Importantly, in vaccinia-A2-G-immunized animals, suppression of homologous challenge virus replication was significantly greater (13-fold) than that observed for the heterologous virus.     

Sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the S, 3, and 3-1 genes.

Four new porcine respiratory coronavirus (PRCV) isolates were genetically characterized. Subgenomic mRNA patterns and the nucleotide sequences of the 5' ends of the S genes, the open reading frame (ORF) 3/3a genes, and the ORF 3-1/3b genes of these PRCV isolates were determined and compared with those of other PRCV and transmissible gastroenteritis virus (TGEV) isolates. The S, ORF 3/3a, and ORF 3-1/3b genes are under intense study because of their possible roles in determining tissue tropism and virulence. Northern (RNA) blot analysis of subgenomic mRNAs revealed that mRNA 2, which encodes for the S gene, of the PRCV isolates migrated faster than the mRNA 2 of TGEV. The PRCV isolates AR310 and LEPP produced eight subgenomic mRNA species, the same number as produced by the virulent Miller strain of TGEV. However, the PRCV isolates IA1894 and ISU-1 produced only seven subgenomic mRNA species. All four of the PRCV isolates were found to have a large in-frame deletion in the 5' end of the S gene; however, the size and location of the deletion varied. Analysis of the ORF 3/3a gene nucleotide sequences from the four PRCV isolates also showed a high degree of variability in this area. The ORF 3 gene of the PRCV isolates AR310 and LEPP was preceded by a CTAAAC leader RNA-binding site, and the ORF 3 gene was predicted to yield a protein of 72 amino acids, the same size as that of the virulent Miller strain of TGEV. The PRCV isolates AR310 and LEPP are the first PRCV isolates found to have an intact ORF 3 gene. The ORF 3a gene of the PRCV isolate IA1894 was preceded by a CTAAAC leader RNA-binding site and was predicted to yield a truncated protein of 54 amino acids due to a 23-nucleotide deletion. The CTAAAC leader RNA-binding site and ATG start codon of ORF 3 gene of the PRCV isolate ISU-1 were removed because of a 168-nucleotide deletion. Analysis of the ORF 3-1/3b gene nucleotide sequences from the four PRCV nucleotides isolates also showed variability.     

Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion.

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37 degrees C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37 degrees C (half-life, approximately 24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37 degrees C (half-life, approximately 30 min). Virions treated at pH 8.0 and 37 degrees C formed clumps and large aggregates. With virions treated at pH 8.0 and 37 degrees C, the amino-terminal peptide E2N (or S1) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37 degrees C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37 degrees C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2C (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37 degrees C or following treatment with guanidine and urea at pH 6.0 and 37 degrees C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37 degrees C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles.     

Intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence.

Cats infected with virulent feline coronavirus strains develop feline infectious peritonitis, an invariably fatal, immunologically mediated disease; avirulent strains cause either clinically inapparent infection or mild enteritis. Four virulent coronavirus isolates and five avirulent isolates were assessed by immunofluorescence and virus titration for their ability to infect and replicate in feline peritoneal macrophages in vitro. The avirulent coronaviruses infected fewer macrophages, produced lower virus titers, were less able to sustain viral replication, and spread less efficiently to other susceptible macrophages than the virulent coronaviruses. Thus, the intrinsic resistance of feline macrophages may play a pivotal role in the outcome of coronavirus infection in vivo.     

Defective endogenous proviruses are expressed in feline lymphoid cells: evidence for a role in natural resistance to subgroup B feline leukemia viruses.

Endogenous feline leukemia virus (FeLV)-related sequences (enFeLV) are a family of proviral elements found in domestic cats and their close relatives. These elements can recombine with exogenous, infectious FeLVs of subgroup A (FeLV-A), giving rise to host range variants of FeLV-B. We found that a subset of defective enFeLV proviruses is highly expressed in lymphoma cell lines and in a variety of primary tissues, including lymphoid tissues from healthy specific-pathogen-free cats. At least two RNA species were detected, a 4.5-kb RNA containing gag, env, and long terminal repeat sequences and a 2-kb RNA containing env and long terminal repeat sequences. Cloning of enFeLV cDNA from two FeLV-free lymphoma cell lines (3201 and MCC) revealed a long open reading frame (ORF) encoding a truncated env gene product corresponding to the N-terminal portion of gp70env. Interestingly, all of three natural FeLV-B isolates include 3' env sequences which are missing from the highly transcribed subset and hence must be derived from other enFeLV elements. The enFeLV env ORF cDNA clones were closely similar to a previously characterized enFeLV provirus, CFE-16, but were polymorphic at a site corresponding to an exogenous FeLV neutralization epitope. Site-specific antiserum raised to a C-terminal 30-amino-acid peptide of the enFeLV env ORF detected an intracellular product of 35 kDa which was also shed from cells in stable form. Expression of the 35-kDa protein correlated with enFeLV RNA levels and was negatively correlated with susceptibility to infection with FeLV-B. Cell culture supernatant containing the 35-kDa protein specifically blocked infection of permissive fibroblast cells with FeLV-B isolates. We suggest that the truncated env protein mediates resistance by receptor blockade and that this form of enFeLV expression mediates the natural resistance of cats to infection with FeLV-B in the absence of FeLV-A.     

Antigenic composition of different forms of Sh. sonnei]

Data are presented on the study, with the aid of immunophoresis, of the antigenic composition of the I, II phases and R-forms of Sh. sonnei. From 5 to 7 antigens differing by thermoresistance, electrophoretic and diffuse mobility were found in the composition of microbial cells of various forms Sh. sonnei. Differences between the I phase on the one hand and cells of the II phase and R-form, on the other hand, consisted both in the quantitative composition of components of their thermostable O-antigens and in the structure of specific lipopolysaccharides of the forms.     

Xanthine oxidase activities: evidence for two catalytically different types.

Xanthine dehydrogenase (EC 1.2.1.37) from mouse small intestine was accompanied by 20% as much xanthine oxidase (EC 1.2.3.2) activity (dehydrogenase-associated oxidase). NAD⁺ and oxygen did not compete as electron acceptors. Upon incubation at 37 °C, the dehydrogenase activity was gradually transformed to oxidase activity. Unexpectedly, the oxidase thus formed (dehydorgenase-derived oxidase) had catalytic properties different from those of the dehydrogenase-associated oxidase. The activation energy for the dehydrogenase-associated oxidase was 20,600 cal/mol, whereas that for the dehydrogenase-derived oxidase was 13,500 cal/mol. The activation energy for the dehydrogenase was 14,000 cal/mol. Between pH 6.4 and 8.5, the activity of the dehydrogenase-associated oxidase was essentially pH independent, whereas the activities of the dehydrogenase-derived oxidase and the dehydrogenase were enhanced with increasing pH. Use of the transformation inhibitor, dithiothreitol, and the protease inhibitor, diisopropylfluorophosphate, showed that these catalytic differences were not the result of partial proteolysis of the enzyme. The data demonstrate the existence of two catalytically different types of mammalian xanthine oxidase activities: A dehydrogenase-associated oxidase and a dehydrogenase-derived oxidase.     

Serum neutralization of feline immunodeficiency virus is markedly dependent on passage history of the virus and host system.

Sera from feline immunodeficiency virus (FIV)-infected cats exhibited extremely low levels of neutralizing antibodies against virus passaged a few times in vitro (low passage), when residual infectivity was assayed in the CD3+ CD4- CD8- MBM lymphoid cell line or mitogen-activated peripheral blood mononuclear cells. By sharp contrast, elevated titers of highly efficient neutralizing activity against FIV were measured, by use of high-passage virus, in assays on either the fibroblastoid CrFK or MBM cell line. However, high-passage virus behaved the same as low-passage virus after one in vivo passage in a specific-pathogen-free cat and reisolation. Subneutralizing concentrations of infected cat sera enhanced the production of low-passage virus by MBM cells, an effect not seen with high-passage virus in CrFK cells. These qualitative and quantitative discrepancies could not be attributed to differences in the amount of immunoreactive viral material, to the amount of infectious virus present in the viral stocks, or to the presence of anti-cell antibodies. The observed effects were most likely due to the different passage history of the viral preparations used. The observation that neutralizing antibodies detected with high-passage virus were broadly cross-reactive in assays with CrFK cells but isolate specific in MBM cells suggests also that the cell substrate can influence the result of FIV neutralization assays. This possibility could not be tested directly because FIV adapted to grow in CrFK cells had little infectivity for lymphoid cells and vice versa. In vitro exposure to infected cat sera had little or no effect on the ability of in vivo-passaged FIV to infect cats. These data reveal no obvious relationship between titers against high-passage virus and ability to block infectivity of FIV in cats and suggest caution in the use of such assays to measure vaccine efficacy. In conclusion, by contrast with what has been previously reported for the use of CrFK cells and high-passage virus, both natural and experimental infections of cats with FIV generate poor neutralizing antibody responses with regard to in vivo protection.