Immunofluorescence of green monkey kidney cells infected with adenovirus 12 and with adenovirus 12 plus simian virus 40

Malmgren, Richard A. (National Cancer Institute; Bethesda, Md.), Alan S. Rabson, Paula G. Carney, and Frances J. Paul. Immunofluorescence of green monkey kidney cells infected with adenovirus 12 and with adenovirus 12 plus simian virus 40. J. Bacteriol. 91:262-265. 1966.-Immunofluorescence studies of the viral antigens and tumor (T) antigens of adenovirus 12 and simian virus 40 (SV40) in green monkey kidney (GMK) cells infected with adenovirus 12 alone or in combination with the SV40 virus showed that the adenovirus 12 viral antigen was produced in detectable amounts only in the cells infected with both viruses. The adenovirus 12 T antigen, on the other hand, was formed in the GMK cells infected with the adenovirus 12 only. This antigen was formed as early as 18 hr after viral infection, and persisted for at least 48 hr after virus infection. There was a correlation between the appearance of the immunofluorescent T antigen in the nucleus and the electron microscope appearance of "nuclear stippling," which developed in the nuclei of GMK cells after infection with adenovirus 12 only, as well as after infection with both viruses.     

Intracellular development of membrane protein of influenza virus.

The intracellular development of membrane protein (MP) of influenza A virus was investigated by immunofluorescent staining. Monospecific antiserum was prepared by immunizing rabbits with MP eluted from SDS-polyacrylamide gels of SDS-disrupted NWS virions. In the productive infection in clone 1-5C-4 cells, MP antigen was first detected over the whole cell at 4 hr after infection, concomitantly with the appearance of hemagglutinin (HA) antigen in the cytoplasm, and bright nuclear fluorescence was then observed. Nucleoprotein (NP) antigen was detected in the nucleus prior to the appearance of fluorescence of MP antigen and thereafter the cytoplasmic fluorescence developed. Late in infection, all of these three antigens were observed predominantly in the cytoplasm with stronger fluorescence at the cell surface. Essentially similar findings were obtained in the abortive infections in L cells and BHK cells. The above results suggest that the membrane protein of influenza A virus is present in the nucleus as well as in the cytoplasm of infected cells.     

Importance of calcium-binding site 2 in simian virus 40 infection

The exposure of molecular signals for simian virus 40 (SV40) cell entry and nuclear entry has been postulated to involve calcium coordination at two sites on the capsid made of Vp1. The role of calcium-binding site 2 in SV40 infection was examined by analyzing four single mutants of site 2, the Glu160Lys, Glu160Arg, Glu157Lys (E157K), and Glu157Arg mutants, and an E157K-E330K combination mutant. The last three mutants were nonviable. All mutants replicated viral DNA normally, and all except the last two produced particles containing all three capsid proteins and viral DNA. The defect of the site 1-site 2 E157K-E330K double mutant implies that at least one of the sites is required for particle assembly in vivo. The nonviable E157K particles, about 10% larger in diameter than the wild type, were able to enter cells but did not lead to T-antigen expression. Cell-internalized E157K DNA effectively coimmunoprecipitated with anti-Vp1 antibody, but little of the DNA did so with anti-Vp3 antibody, and none was detected in anti-importin immunoprecipitate. Yet, a substantial amount of Vp3 was present in anti-Vp1 immune complexes, suggesting that internalized E157K particles are ineffective at exposing Vp3. Our data show that E157K mutant infection is blocked at a stage prior to the interaction of the Vp3 nuclear localization signal with importins, consistent with a role for calcium-binding site 2 in postentry steps leading to the nuclear import of the infecting SV40.     

Replication of human immunodeficiency virus in two T-cell lines and their application as antigens for immunofluorescence

Two T-cell lines, TALL-1 and CCRF-CEM, were infected with human immunodeficiency virus (HIV), strain LAV, to explore the time course of the appearance of various virus specific antigens, and to establish an antibody assay system by indirect immunofluorescence (IF). These cells were infected with LAV at two different input multiplicity of infection (MOI). Antigens were tested by Western blot analysis (WB) and IF. Antigens for WB were extracted from the infected cells at various times after infection, but pooled sera of American HIV carriers could not recognize gp41 or gp160. Antigen expression was highest in CCRF-CEM, but, as the antigen for IF, TALL-1 infected at the MOI of 8.0 was the most suitable 7 days after infection, because it includes a fairly large number of uninfected cells, which served as the internal control.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

Induction of Epstein-Barr virus nuclear antigen and DNA synthesis in a human epithelial cell line after Epstein-Barr virus infection.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma is supported by the presence of EBV genomes in the epithelial elements of the tumor and by elevated antibody titers to EBV-specific antigens in the patients; the levels of these titers are related to the clinical course of the disease. However, since most laboratory data suggest that EBV is a B-lymphotropic virus, it is unclear how the virus becomes associated with the epithelial elements of the nasopharynx. The purpose of the present work was to find a human model system to study this association. A human epithelial line (U) was found that could be directly infected by EBV, and viral functions, the induction of EBV nuclear antigen and cellular DNA synthesis, were demonstrated. The U line was established in 1957 by the late H. J. Van Kooten (Kok-Doorschodt at the University of Utrecht), and although it is no longer diploid, it exhibits density inhibition. When U cells were infected with EBV, EBV nuclear antigen was expressed in 6 to 16% of the cells, 1 and 2 days after infection with B95-8 virus, but not with the P3HR-1 strain. No evidence for virus replication was obtained; immunofluorescence staining for early antigens and virus capsid antigens gave negative results. Quantitative adsorption experiments for EBV indicated that the adsorption capacity of U cells is significant (60% of Raji cells). The present results also demonstrated that infection with the virus overcomes block(s) in cellular DNA synthesis caused by 5-fluorodeoxyuridine. The induction of DNA synthesis was determined by increased incorporation of [3H]thymidine into the cells. The highest level of isotope incorporation was observed at about 15 h after infection and thereafter decreased. Analysis of the induced DNA indicated that it was of cellular origin.     

Cooperation of structural proteins during late events in the life cycle of polyomavirus.

The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1.     

Cytoplasmic inclusions in canine cells infected with infectious canine laryngotracheitis (ICL) adenovirus.

Spherical dark inclusions were observed in both the cytoplasm and nuclei of infectious canine laryngotracheitis (ICL) adenovirus infected MDCK (Madin-Darby Canine Kidney) cells. The distribution of these inclusions in the cells appeared to indicate that they were formed in both the cytoplasm and in the nucleus at about the same time and there did not appear to be movement of these inclusions between the cytoplasm and the nucleus during the early stages of infection. Morphological appearance, 3H-leucine autoradiography, and immunoferritin labelling showed that the cytoplasmic inclusions were similar in nature to the dark nuclear inclusions, and contained the adenovirus hexon antigen, but not the penton base and fiber antigens.     

Presence of herpes simplex virus-related antigens in transformed L cells.

Antiserum prepared against herpes simplex virus type 1 (HSV-1)-infected L cells, i.e., lytic antiserum, was shown by an indirect immunofluorescence test to stain 90 percent of HSV-transformed L or HeLa cells. Immunofluorescence in these cells was always most intense in the perinuclear cytoplasmic region. Similar results were obtained with antiserum prepared against HSV-transformed L cells. These data indicate that HSV-transformed cells (both L and HeLa) express HSV-related antigens. Antiserum prepared against HSV-1-transformed L cells, i.e., transformed-cell antiserum, was found to agglutinate purified HSV type 1 virions but failed to neutralize infectivity. This suggests that HSV-1 structural antigens are expressed in HSV-1-transformed L cells. Immunodiffusion studies showed that at least two HSV-related antigens could be demonstrated with antigens from HSV-1-transformed L cells and transformed-cell antiserum. These two antigens were shown to be present in all clonal lines of HSV-1-transformed cells examined, six L cell lines and one HeLa cell line. Therefore, we conclude that transformation of cells by HSV-1, which is known to be associated with acquisition of viral thymidine kinase, must also be associated with the presence of these two antigens. We performed experiments showing that there are species of HSV-related antibody in HSV-transformed cell antiserum that could not be absorbed out with antigens from HSV-infected L cells. Antibodies present in lytic antiserum were completely removed by antigen preparations from cells lytically infected with HSV-1. Also, lytic antiserum failed to block HSV-related staining of transformed L cells in a direct immunofluorescence test. These results are compatible with one of two notions: either (i) certain genes are expressed during transformation that are not expressed during lytic infection, or (ii) these genes are expressed to a much more reduced extent during lytic infection than in transformed cells.     

Immunofluorescence and Cytochemical Studies of Visna Virus in Cell Culture

Sequential morphological changes occurring in sheep choroid plexus cells infected with visna virus were studied by direct immunofluorescence, acridine orange, and hematoxylin and eosin staining methods. Specific immunofluorescence was first detected in the perinuclear cytoplasm of solitary cells 24 hr after infection. As the infection progressed, viral antigen appeared in an increasing number of cells, and rounded globular cells with long slender processes harboring intense fluorescence were seen. Nuclear fluorescence was not observed in infected monolayers. Polykaryocytes formed within 6 hr after inoculation due to the direct cell-fusing effect of the virus inoculum did not show specific fluorescence. Viral antigen was found, however, in the cytoplasm of multinucleated giant cells in cover slips harvested after new infective virus had been released, and later in the course of infection circular fluorescent inclusions were seen in the cytoplasm of polykaryocytes. Comparable eosinophilic inclusions were observed in hematoxylin and eosin preparations, and acridine orange staining of infected monolayers demonstrated similar inclusions which fluoresced with the color characteristic of single-stranded nucleic acid and were susceptible to digestion with ribonuclease. Visna virus appears to be a ribonucleic acid virus which replicates in the cytoplasm.