Identification of the most significant amphipathic helix with application to HIV and MHV envelope proteins.

Amphipathic helices, which play important roles in protein structure, occur in a wide variety of lengths. Yet existing methods employ fixed window lengths. We present a hierarchical procedure that identifies the Q most significant amphipathic helices regardless of length. Since the observed hydrophobicities are not normally distributed, test statistics usually employed for least-squares regression are inappropriate for assessing statistical significance of amphipathic helices. We show that an adjusted F statistic provides a good test. An application to the envelope protein of HIV finds an unexpected long amphipathic helix in gp41.

Monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions.

Monoclonal antibodies (MAbs) directed against the E2 glycoprotein of mouse hepatitis virus (MHV) have been classified according to their ability to bind to either of the two purified 90,000-molecular-weight subunits (90K subunits) of the 180K peplomeric glycoprotein E2. Correlation with previously reported information about these MAbs suggest that both of the subunits of E2 are important for viral infectivity and cell fusion. Incubation of trypsin-treated virions at pH 8.0 and 37 degrees C released only the E2N subunit from virions. The pattern of MAb reactions suggested that a conformational change occurred in the E2N subunit in association with its release from virions under mildly alkaline conditions at 37 degrees C, the same conditions which are optimal for coronavirus-induced cell fusion.

Measurement of the concentration of murine IgG monoclonal antibody in hybridoma supernatants and ascites in absolute units by sensitive and reliable enzyme-linked immunosorbent assays (ELISA).

We have investigated an enzyme-linked immunosorbent assay (ELISA) for mouse IgG using affinity-purified goat anti-mouse antibodies for capture and detection. This assay was used to measure the absolute or weight/volume concentration of murine monoclonal antibody in hybridoma supernatants. Bovine or subclasses except IgG3 in the 1-20 ng/ml range. Antibody capture was essentially complete in the optimized assay. In combination with an antigen-dependent ELISA, the assay allowed estimation of the absolute concentration of specific monoclonal antibody in ascites. These rapid and relatively simple assays may be applicable in many situations in which a practical means of measuring murine monoclonal antibodies in weight/volume units is needed.

Analysis of JHM central nervous system infections in rats.

Intracerebral inoculation of murine coronavirus JHM into 2- to 3-day-old Wistar Furth rats causes an acute encephalomyelitis, while inoculations at 10 days of age usually result in hind leg paralysis. To examine the distribution of viral antigens within this infected central nervous system (CNS) tissue, we used the avidin-biotin-peroxidase method to detect monoclonal and polyclonal antibodies bound to JHM structural proteins; in addition we used the Western blot technique to detect viral proteins. Our study demonstrated the following characteristics: Infected neuronal and glial cells produced viral nucleocapsid and E2 glycoprotein. The synthesis of these viral structural proteins was not restricted to cells in any particular part of the central nervous system. While JHM E2 proteins could be detected in individual cells of JHM-infected CNS tissue, the relative level of detectable E2 protein in the total CNS tissue of infected rats was reduced by more than 13-fold compared with JHM-infected tissue culture cells. Hippocampus neuronal cells provided a sensitive indication of JHM infection. These cells invariably contained antigens in both acutely and chronically infected animals. The distribution of cells containing viral antigens differed markedly for JHM-induced acute encephalitis and chronic demyelinating disease. Acutely infected brains had large lesions containing low levels of viral antigen scattered throughout the brain. One percent to ten percent of histologically normal cells in many parts of the brain contained viral antigens; in addition, more neuronal cells than glial cells were observed to be antigen-positive. The hippocampus appeared normal with hematoxylin-eosin staining; however, a scattered infection of neuronal cells was apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.

The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1.

Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.

Presence of leader sequences in the mRNA of mouse hepatitis virus.

To determine the structure and the mechanism of synthesis of mouse hepatitis virus mRNA, the map positions of the large RNase T1-resistant oligonucleotides of the seven mouse hepatitis virus strain A59 intracellular mRNA species were studied. We found that all but one of the oligonucleotides were mapped at the positions within each mRNA consistent with the nested-set, stairlike structure of mouse hepatitis virus mRNA (Lai et al., J. Virol. 39:823-834). However, one oligonucleotide, 10, was mapped near the 5' ends of every mRNA and virion genomic RNA. In other words, oligonucleotide 10 and, therefore, the sequences around the 5' ends of the mRNAs are not colinear with the genomic sequences. Because this oligonucleotide is present only once in the genomic RNA, this result indicates that oligonucleotide 10 is not transcribed from multiple sites on the genomic template, but rather represents a leader RNA sequence which is joined to the body sequences of the different mRNAs during mRNA transcription. This provides the most direct evidence thus far for the presence of leader sequences in the mRNAs of mouse hepatitis virus, which is a cytoplasmic virus. Several possible mechanisms of RNA synthesis are discussed.

Characterization of small plaque mutants of mouse hepatitis virus, JHM strain.

Two small plaque mutants designated as 1a and 2c were isolated from DBT cells persistently infected with the JHM strain of mouse hepatitis virus. Unlike the wild type JHM, these two mutant viruses grew more slowly with no prominent cell fusion. The buoyant densities of the mutants were slightly lower and 2c was revealed to have fewer peplomers than JHM by electron microscopy. The purified JHM contained five polypeptides with molecular weights (M.W.) of 260,000, 105,000 (GP105), 65,000, 60,000 (P60), and 23,000 (GP23). In addition to two polypeptides, P60 and GP23, which were common to JHM and the mutants, 1a was found to contain three other specific polypeptides with M.W. of 180,000 (GP180), 110,000, and 95,000 (GP95), while 2c had GP180, GP105, GP95, and one with a M.W. of 175,000. All of these polypeptides were shown to be glycosylated except for P60. After bromelain treatment, all these viruses lost the peplomers and contained P60 and another new 18,000 dalton polypeptide.

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.

The polypeptides of human and mouse coronaviruses. Brief report.

The polypeptide compositions of two coronaviruses, human coronavirus strain 229E (HCV229E) and mouse hepatitis virus strain 3 (MHV3), were characterised on polyacrylamide gels. Similar polypeptide patterns were observed for both viruses consisting of large surface projection glycopolypeptides of mol. wt. 160,000 and 105,000 for HCV229E, and 170,000 and 90,000 for MHV3, two small polypeptides of mol. wt. varying from 24,000 to 20,000, and a polypeptide of mol. wt. 50,000. The results are discussed with respect to previous reports of the polypeptides of these and other coronaviruses.

Phosphoproteins of murine hepatitis viruses.

Four strains of the coronavirus murine hepatitis virus were examined for the presence of phosphorylated proteins. The nucleocapsid protein was determined to contain phosphate covalently linked to serine but not to threonine residues. The nucleocapsid protein was the only phosphorylated protein detected in these strains of murine hepatitis virus.

Genomic RNA of the murine coronavirus JHM.

Genomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5.4 to 6.5 X 10(6), as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-)dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.

RNA of mouse hepatitis virus.

The RNA of mouse hepatitis virus, a coronavirus, was isolated from the virus released early in the infection and analyzed by sucrose gradient sedimentation and electrophoresis. It was found to consist of a piece of single-stranded RNA of about 60S. Its molecular weight was estimated to be 5.4 X 10(6) by electrophoresis in methylmercury-agarose gels. At least one third of the RNA contained polyadenylated sequences. It is, therefore, probably positive stranded. The virus harvested late in the infection contained, in addition to 60S, some 30 to 50S RNA that are possibly degradation products of the 60S RNA. No difference in the electrophoretic behavior could be detected between the RNA isolated from a pathogenic (JHM) and a nonpathogenic (A59) strain.

In vitro growth characteristics and heterogeneity of mouse hepatitis virus type 3.

The in vitro virus yield of MHV3 reached 10(7) PFU/ml in mouse DBT cells infected with a virus suspension in HEPES-buffered medium containing DEAE-dextran. The virus titer was 10(6) PFU/ml in the presence of 10 micrograms actinomycin D/ml MHV3 growth in DBT cells gave three peaks of density (1.10--1.14 g/cm3, 1.18--1.20 g/cm3, and 1.25--1.31 g/cm3) in sucrose gradients. All these peaks retained infectivity.

Polyadenylate in the virion RNA of mouse hepatitis virus.

Mouse hepatitis (MH) virus was grown in SR-CDF1-DBT, a mouse cell line, and purified by ammonium sulfate precipitation and by density gradient centrifugation. Extraction of RNA from purified virions with 1% SDS and sedimentation analysis of the RNA revealed a major 50S component and two minor components. Treatment of virions with phenol/chloroform also produced the 50S component, although its yield was lower. MH virion RNA can bind to a poly(U)-fiberglass filter, indicating that MH virion RNA contains poly(A). A poly(A)-like fragment was isolated by digestion with ribonuclease A [EC 3.1.4.22] and T1 [EC 3.1.4.8] and by DEAE-Sephadex column chromatography. Analysis of the fragment for base composition showed it to be an adenine-rich material. Its chain length was about 90 nucleotides, as determined by ion-exchange chromatography and gel electrophoresis.