Effect of arginine deficiency on the reproduction of human cytomegalovirus.

In arginine-deprived human embryonic fibroblasts the reproduction cycle of human cytomegalovirus (CMV) is incomplete. Infectious virus cannot be demonstrated in cell disintergrates, and from among the CMV antigens only the diffuse cytoplasmic antigen is detectable by immunofluorescence. The antigens localized in the cell membrane and those appearing during the complete cycle as large granules or inclusion-like bodies in the nucleus do not appear in the absence of arginine. The CMV genome persists in the arginine-deprived culture; after re-feeding with arginine-containing medium, maturation of virions soon ensues. Maturation could be prevented by inhibitors of protein synthesis, but not by DNA inhibitors, added simultaneously with completion of the medium.     

THE LOCALIZATION OF ALBUMIN AND FIBRINOGEN IN HUMAN LIVER CELLS

Human liver sections were stained with anti-human serum albumin and/or anti-human fibrin monomer fluorescent conjugates. Approximately 10 per cent of the hepatic cells stained specifically for human serum albumin,1 per cent for fibrinogen, and 0.1 per cent for both. Approximately 18 per cent of the Kupffer cells stained specifically for human serum albumin and 33 per cent for fibrinogen. Staining of both cell types was mainly cytoplasmic, although albumin was found in the nuclei of some parenchymal cells, depending on the method of fixation. Cytoplasmic granules staining specifically for fibrinogen could be seen just inside the cell membrane facing the bile caniculi in many more parenchymal cells than the 1 per cent showing diffuse cytoplasmic staining. The technical difficulties involved in preparing fluorescent conjugates against these antigens and in the fixation of these antigens for immunofluorescent staining are discussed.     

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.     

Combined Fluorescent-Antibody and Electron Microscopy Study of Marek''s Disease Virus-Infected Cell Culture

Duck embryo fibroblast (DEF) and chicken embryo fibroblast (CEF) cultures infected with Marek's disease virus were studied by combined fluorescent antibody and electron microscopy techniques. In both DEF and CEF cultures, cells containing immunofluorescent (IF) antigen also contained herpesvirus particles; conversely, cells lacking this antigen lacked herpesvirus particles. Two morphologically distinct IF antigens were detected in the cytoplasm. (i) A granular antigen in the perinuclear region was brightly stained with the conjugated antibody. This antigen was composed of a granular mass of osmiophilic material and did not contain virions. (ii) A diffuse antigen, present throughout the cytoplasm of infected cells, was less brightly stained. The area of the cell with the highest concentration of this antigen contained small vesicles, folded membranes, and fine electron-dense granules. Naked virions were occasionally seen in these areas. A diffuse nuclear IF antigen was occasionally seen in infected cells. This antigen was often separated from the nuclear membrane and the nucleolus by a clear margin. The intranuclear IF antigen was composed of a fine granular aggregate and naked herpesvirus particles which were randomly distributed throughout the nucleus. Viral capsids in antibody-treated cells were coated with fine filamentous material.     

免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金所特异标记。本文结果表明,结合于核孔复合体上     

免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体-蛋白A-胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体-蛋白A-胶体金所特异标记。本文结果表明,结合于核孔复合体上的胞质纤维是波形纤维,并且提示波形纤维可能通过与Nup 180的结合附着在核孔复合体上,为进一步探讨中间纤维与核孔运输的关系提供了初步实验证据。     

Localization of a low molecular weight antigen of Eimeria tenella by use of hybridoma antibodies.

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.     

Trypanosoma cruzi: expression of antigens on the membrane surface of parasitized cells

A direct immunofluorescent antibody test with an anti-Trypanosoma cruzi F(ab')2 conjugate was used to demonstrate antigens of T. cruzi on the membrane surface of intact live or fixed macrophages and L929 mouse fibroblasts infected with the organism. Antigens were demonstrated in 5 to 50% of infected cells, and their presence was not directly related to the number of intracellular organisms. Cells with as few as four intracellular amastigotes had demonstrable surface antigens, whereas some cells with as many as twelve or more organisms did not. Capping of antigen-antibody complexes was noted to begin a few minutes after the addition of the anti-T cruzi F(ab')2 conjugate; by 30 min, most of the parasitized cells had eliminated the complexes, and no surface antigen of parasitic nature could be demonstrated. Although capping may have caused a negative result in a previously positive cell, other mechanisms may be involved, because antigens were not demonstrated in some heavily parasitized cells examined immediately after completion of the test. Treatment of the infected cells with trypsin or chymotrypsin resulted in the absence of demonstrable parasite antigens on the cell membrane surface. However, the antigens were again demonstrated 12 hr after the enzymes were removed. The reappearance of parasite antigens on the surface of infected cells was prevented by treatment of the monolayers with puromycin or tunicamycin. A T cell-enriched population of spleen lymphocytes from mice chronically infected with T. cruzi recognized the membrane-bound antigens and proceeded to destroy the host cell and the intracellular organisms. In this process, noninfected cells were also destroyed, possibly because they were coated with antigens released from intact infected cells or from infected cells that had been lysed by the action of the sensitized lymphocytes or their products.     

Mechanism of Intranuclear Crystal Formation of Herpes Simplex Virus as Revealed by the Negative Staining of Thin Sections

Structural alterations induced in HeLa cells by herpes simplex virus and the mechanism whereby the virus is formed in the nucleus in crystal arrays were studied by electron microscopy with both the usual and negatively stained sections. Aggregates of granular and filamentous material were observed in the cytoplasm of infected cells with both sections. On the other hand, no remarkable alterations in appearance of the cytoplasmic ground substance were observed with the usual sections of infected cells. However, the cytoplasmic ground substance of infected cells when negatively stained consisted of granular material which was different in appearance from the spongy material constituting the cytoplasmic matrix of uninfected cells. In the nucleus of infected cells, complexes consisting of round bodies, amorphous material, aggregates of uniform granules in rows, and viral crystals were often observed near the nuclear membrane in both types of sections. Examinations of the granular aggregates with negatively stained sections suggested that each granule represents a subunit and that the several adjoining subunits (approximately eight) constitute the requirement for formation of a single viral capsid with a core. Thus, rapid and simultaneous formation of the core and capsid within the aggregate would replace the rows of the granules with the viral crystal. The advantages of negative staining of thin sections for visualization of fine structural alterations are discussed.     

STUDIES ON ISOLATED MEMBRANES OF AZUROPHIL AND SPECIFIC GRANULES FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

Membranes were prepared from rabbit polymorphonuclear leukocyte azurophil and specific granules separated by zonal differential centrifugation. The two types of granule membranes were quite similar in ultrastructural appearance, but they showed distinct differences in cholesterol-phospholipid ratios and in protein components demonstrable in polyacrylamide gels.     

Cytomegalovirus-induced membrane antigens in productively infected cells.

Adsorbed but not penetrated virus can be removed from the CMV-infected cell membrane by digestion with cystine-activated papain. Membrane antigens appear on 80-90% of the infected cells 14-20 hr after infection as a result of de novo protein synthesis. Antigen synthesis can be blocked with inhibitors of protein synthesis, but not with DNA inhibitors. In the early stage of infection, pooled human convalescent serum reacted well with the membrane antigen, whereas pooled antiserum of rabbits immunized with CMV virion suspension gave a positive reaction with a small proportion of the cells. After the 48th hr, both the human and the rabbit serum pool reacted with the membrane of the infected cells. Absorption with cell cultured for 24 hr after CMV infection reduced the neutralization titres of the antisera only slightly but the titre reduction was considerable when absorption was performed with cells cultured for more than 48 hr after infection. It is concluded that on the membrane of cells productively infected by CMV at least two membrane antigens are present, one coded for by the DNA of the parent virus and another which is the product of the DNA of the virus progeny. The two antigens can be differentiated serologically.     

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.     

Nonuniform distribution of concanavalin-A receptors and surface antigens on uropod-forming thymocytes

Uropods can form spontaneously in a variable fraction of mouse thymocytes incubated for 30--60 min in vitro at temperatures between about 8 degrees and 37 degrees C. The majority of the cells with a typical uropod are medium and large thymocytes. The "normal" distribution of concanavalin-A receptors and antigens recognized by a rabbit anti-mouse thymocyte serum was studied on these cells by electron microscopy using ferritin-conjugated lectin or antibodies. The cells were fixed with glutaraldehyde or formaldehyde before labeling. The distribution was essentially uniform on spherical cells. On the contrary, on cells which had formed a uropod the labeled receptors and antigens appeared to be preferentially concentrated around the nucleus, and depleted over the uropod, and especially over the constriction at the base of the uropod. Uropod formation and inhomogeneous distribution were inhibited or reversed by cytochalasin B, but not by vinblastine or colchicine. When the same ligands were applied to unfixed cells, the labeled and cross-linked components capped normally towards the cytoplasmic pole of the cell. These observations are described in relation to the ability of receptors and antigens to interact with an intracellular mechanical structure, and to the mechanism of capping.     

Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.

The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.     

Immunofluorescent Cell-Counting Assay for Lymphocytic Choriomeningitis Virus

A quantitative assay for lymphocytic choriomeningitis virus was developed and standardized. The assay is based on direct immunofluorescent staining of infected L-929 cell monolayers and enumeration of cells containing fluorescent viral antigens. Maximal adsorption of virus to cells occurred within 1 h. Observations on the sequential development of viral antigens within cells showed that specific cytoplasmic fluorescence appeared within 10 h. The optimal time for enumerating fluorescent cells was from 18 to 20 h after addition of virus. A linear relationship was demonstrated between the number of infected cells and the relative virus concentration. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The immunofluorescent cell-counting assay for lymphocytic choriomeningitis virus was highly precise and reproducible.     

Associations of PKC isoforms with the cytoskeleton of B16F10 melanoma cells.

Although PKC plays a major role in regulating the morphology and function of the cytoskeleton, little is known about in situ associations of specific isoforms with the cytoskeleton. We demonstrate that seven PKC isoforms are expressed in B16F10 melanoma cells and show different levels of induction by serum. Using cell cytoskeleton preparations (CSKs), confocal microscopy, and immunocytochemistry, all isoforms show specific patterns of localization to focal contact-like structures (alpha, delta), very small cytoplasmic granules/vesicles (all isoforms), dense ordered arrays of small granules in the perinuclear region (alpha, delta), granules/vesicles associated with a homogeneous framework in the cytoplasm adjacent to the nucleus (gamma), or irregular-shaped patches of granules at or near the nuclear perimeter (eta, theta). In addition, several isoforms are present as cytoplasmic granules/ vesicles in linear or curvilinear arrays (alpha, delta, epsilon, theta). When isoform localization is examined using 3.7% formaldehyde or methanol:acetone, the patterns of localization in CSKs are often difficult or impossible to detect, and many are described here for the first time. Double-labeling experiments with CSK demonstrate that PKC actin co-localizes with punctate alpha-rich particles above the nucleus, granules of epsilon throughout the cytoplasm, and with theta in irregular-shaped aggregates associated with the nucleus. Vimentin co-localizes with perinuclear granules of delta and beta(2), and alpha-tubulin co-localizes with theta in structures at or near the nuclear surface and in microtubules associated with the microtubule organizing center (MTOC). In summary, the present study demonstrates that seven PKC isoforms are endogenously expressed in B16F10 melanoma cells. These isoforms show various levels of induction by serum and specific patterns of association with various components of the detergent-resistant cell cytoskeleton.     

Lymphocytes with parallel tubular structures: morphologically a distinctive subpopulation

Peripheral blood lymphocytes were fractionated in T, B and Null cell enriched subsets by means of sheep red blood cell rosette (ESRBC) sedimentation and nylon wool adherence. The ultrastructural features of these subpopulation were investigated. The T cell fraction in which the sheep erythrocytes were removed from the ESRBC rosette-forming cells (ESRBC-RFC) by lysis with ammonium chloride, consisted mainly of two morphologically distinctive subsets. The majority of the cells (80%) displayed a smooth surface membrane and had a high nuclear to cytoplasmic ratio with few cytoplasmic organelles. The other cell type (18%) had a relatively rough surface membrane, a low nuclear to cytoplasmic ratio, often an indented nucleus and numerous cytoplasmic organelles such as characteristic amorphous granules and sometimes parallel tubular structures (p.t.s.). If the T cells were obtained after mechanical vibration of the ESRBC-RFC, the majority of these cells appeared morphologically identical to this latter cell type. Cells with p.t.s. and amorphous granules were also demonstrated within the Null and B cell enriched fractions (50% and 25% respectively), though in the B cell enriched fraction this cell type is probably due to a contamination of Null cells. Previous observations had already demonstrated that these cells in the three fractions represent the Fc gamma receptor-bearing lymphocytes. The similarities suggest that the Fc gamma receptor-bearing and p.t.s. containing lymphocytes form a morphologically distinct subpopulation.     

APPEARANCE AND DISTRIBUTION OF FERRITIN IN MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF HETEROLOGOUS ERYTHROCYTES

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.     

A Comparison of the Killing of Cultured Mammalian Cells Induced by Decay of Incorporated Tritiated Molecules at -196°C

The killing efficiency of tritium disintegrations in frozen mammalian cells labeled with tritiated uridine, histidine, and lysine was compared with the killing efficiency of incorporated tritiated thymidine. In each case, the distribution of tritium in the cells was determined by chemical fractionation as well as by radio-autography. Of all tritium disintegrations, by far the most effective were those occurring in DNA molecules within frozen cells; such incorporated tritium has a killing efficiency of 0.006. When cells were incubated with tritiated uridine for 10 min to label nuclear RNA, the killing efficiency was 0.0015. When the cells were pulse labeled with tritiated uridine and permitted to grow in nonradioactive media for 10 hr before freezing in order to incorporate tritium into cytoplasmic RNA, the killing efficiency was reduced to 0.0010. The results suggest that decay of tritium in nuclear RNA is more effective than that in cytoplasmic RNA. When the cells were labeled with tritiated histidine or lysine for 30 min, tritium atoms were found mainly in the acid soluble rather than in the protein fraction and the killing efficiency in each case was approximately 0.0007. The results of these suicide experiments indicate that the killing efficiency of tritium disintegrations depends on where tritium is located within the cells. Tritium disintegrations in the nucleus are more effective in killing the cell than that in cytoplasm; and tritium disintegrations on DNA in the nucleus is more effective in killing the cell than that of nuclear RNA.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: I. Patterns of Ribonucleic Acid Synthesis in Productively Infected Cells

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4S RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18S and 28S rRNA remained identical and higher than that of 4S RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45S rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28S and 18S rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45S RNA and a decrease in the rate of processing of that 45S RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50S or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16S to 20S, but there is some rapidly sedimenting RNA (> 28S) here too. (iv) Finally, there was leakage of low-molecular weight (4S) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.