Further observations on the morphogenesis of human rotavirus in MA 104 cells

Summary Human rotavirus KUN strain was cultivated in a fetal rhesus monkey kidney cell line, MA 104 cells. Four types of virus particles in cells infected with KUN strain were clearly identified: nucleoid cores, single-shelled particles, double-shelled particles, and membrane band, enveloped particles. Enveloped particles were found only in the thin sections of infected cells. When first visible, the virus precursors appeared at the ribosome free membrane of rough endoplasmic reticulum (RER), increasing in size while simultaneously being coated with nucleocapsid, inner shell. Single-shelled particles were also synthesized within bundles of filaments of viroplasm in the cytoplasma. During subsequent virus maturation two types of budding processes were observed. Double-shelled particles arising at the RER membrane entered the cisternae of the RER through an exocytosis-like process. In contrast, the enveloped particles developed in the cisternae by being completely enclosed with RER membrane, and later during cytolysis released the single-shelled particles. These enveloped virus particles appeared to be the result of inefficient virus maturation at the last stage of outer capsid formation.With 11 Figures     

Defective rotavirus particle assembly in lovastatin-treated MA104 cells

Rotavirus is a non-enveloped virus that depends on cellular lipids for cell entry and associates with lipid rafts during assembly. However, the effects of cellular lipids on rotavirus assembly are still not fully understood. The present study analyzes the effects of lovastatin, an inhibitor of cholesterol biosynthesis, during rotavirus infection in MA104 cells with regard to viral growth and particle assembly. Following viral infection, a 2-log relative reduction of viral titers was observed in drug-treated cells, while viral mRNA levels in infected cells remained unaltered in both groups. Furthermore, the levels of some viral proteins in drug-treated cells were elevated. The observed discordance between the viral RNA and protein levels and the decrease in infectivity titers of viral progeny in the drug-treated cells suggested that the drug affects viral assembly, the viral proteins not being properly incorporated into virions. Transmission electron microscopic (TEM) analysis revealed that in drug-treated cells there was an increase in “empty-looking” rotavirus particles devoid of an electron-dense core as compared to the normal, electron-dense particles seen in untreated infected cells. The present study thus provides visual evidence of defective rotavirus particle assembly as a result of cholesterol depletion. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination policy.     

Two modes of entry of reovirus particles into L cells

Evidence is presented supporting the hypothesis that reovirus intermediate subviral particles (ISVP), which show increased infectivity relative to intact virions, can gain entry into host L cells by two alternative pathways. One pathway is by the process of viropexis, involving phagocytic vacuoles. A second entry pathway is via direct penetration of the plasma membrane of the cell, without involvement of a phagocytic vacuole. Using electron microscopy, a kinetic analysis of the uptake process was carried out. Results indicate that at 37 degrees C ISVP gain entry into host cells primarily by direct entry, although viropexis also occurs, while intact virions gain entry by viropexis almost exclusively. A second line of experimental evidence consistent with the idea that ISVP can 'melt' their way through the plasma membrane is provided by studies on the release of pre-loaded radioactive 51Cr from host cells following infection. 51Cr release data demonstrate that infection with ISVP leads to an immediate increased leakiness of the cell plasma membrane, whereas no such increase takes place following infection with an equivalent number of intact virions. This demonstrates that ISVP can interact with the plasma membrane of the cell in a manner which is qualitatively different from the interaction between intact virions and the plasma membrane. The ability of ISVP to directly penetrate the plasma membrane of the host cell, which intact virions apparently cannot do, could explain the decreased duration of the eclipse phase, as well as the increased infectivity of ISVP, relative to that observed for infection with intact virions.     

Effect of hypertonic conditions on protein synthesis in MA104 cells infected with human rotavirus

When a high NaCl concentration was used to decrease selectively the synthesis of cell proteins, the synthesis of most cellular polypeptides was greatly diminished relative to human rotavirus proteins. Thus, in the presence of 150 mM excess NaCl, 11 viral polypeptides were clearly identified. However, hypertonic conditions also reduced viral protein synthesis to a different extent with individual proteins. No significant changes in viral protein synthesis occurred during incubation under the hypertonic condition for up to 6 h, and infectious virus yields of MA104 cells incubated in the hypertonic medium did not differ from the yields of untreated MA 104 cells. These results indicate that hypertonic conditions provide a useful tool for qualitative studies of viral protein synthesis in human rotavirus infected cells.     

MA104细胞牛轮状病毒受体的提纯及鉴定

目的研究ＭＡ１０４细胞上的牛轮状病毒（ＢＲＶ）受体。方法ＢＲＶ受体的单克隆抗体（单抗）与溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ偶联，利用免疫亲和层析法从ＭＡ１０４细胞膜制备物中提纯了ＢＲＶ受体。结果间接酶联免疫吸附试验（ＥＬＩＳＡ）及免疫斑点法表明提纯物呈ＢＲＶ受体抗原阳性；ＳＤＳ－聚丙烯酰胺凝胶电泳显示单抗偶联柱层析洗脱物仅有单一区带与洗脱峰对应；Ｗｅｓｔｅｒｎｂｌｏｔ表明提纯物能够免被受体单抗识别；固相分析实验表明纯化物具有结合ＢＲＶ的能力；提纯物的小鼠免疫血清可以阻断ＢＲＶ感染宿主细胞。结论纯化物为ＢＲＶ受体     

Further investigation on the mode of entry of human rotavirus into cells

Summary Monoclonal antibodies (MAbs) to 2 epitopes on the haemagglutininneuraminidase (HN) protein of the Ulster strain of Newcastle disease virus neutralized synergistically: MAbs to HN-1 and HN-2 neutralized 1.2 and 1.7 log₁₀ infectious units (i. u.) of virus when single as compared to 4.1 when combined. Although MAb to HN-1 but not to HN-2 inhibited haemagglutination they both neutralized more virus on desialized cells compared to normal cells and were considered to have interfered with viral attachments in a cooperative manner when combined. A third MAb to fusion (F) protein reduced infectivity by five log₁₀ i. u. HN-1, HN-2 and F were not the only immunodominant epitopes because mutants, which simultaneously lacked all 3 epitopes, were indistinguishable from wild type when neutralized by polyclonal mouse, rabbit or chicken antiserum to whole virus.With 2 Figures     

Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2).

The production of viral antigen after infection of MA104, HepG2 (derived from human liver), and CaCo-2 (derived from human colon) cells with various cultivatable human and animal rotavirus strains was compared using immunofluorescence tests. All rotavirus strains examined expressed antigen in CaCo-2 cells and MA104 cells, but only some virus strains, namely, SA11-Cl3 (simian), RRV (simian), CU-1 (canine), and Ty1 (turkey), produced antigen in numbers of infected HepG2 cells comparable to infections in MA104 and CaCo-2 cells. Fl-14 (equine), OSU (porcine), NCDV (bovine), and Ch2 (chicken) strains were found to infect moderate numbers of HepG2 cells. Most human rotaviruses (representing viruses in serotypes 1, 2, 3, 4, 8, and 9), a simian rotavirus variant (SA11-4F), lapine (Ala, C-11 and R-2) viruses and porcine (Gottfried) virus infections resulted either in no detectable antigen or antigen synthesis in a low percentage of HepG2 cells. Human rotavirus isolates obtained from the stool specimens of an immunocompromised child with rotavirus antigen in his liver showed two different patterns of replication in HepG2 cells. Examination of the replication of a subset of viruses in the liver and intestinal tissues of orally infected suckling mice showed the CU-1 and Ty1 strains replicated well, while the OSU and human rotavirus strains did not. These results indicate that growth restriction in HepG2 cells is not serotype-specific, and growth of a virus in HepG2 cells does not necessarily correlate with the hepatotropic potential of a virus strain. Factors that may influence these differences of virus infectivity in HepG2 cells are discussed.     

Monoclonal antipeptide antibodies recognize epitopes upon VP4 and VP7 of simian rotavirus SA11 in infected MA104 cells

Summary To study morphogenetic events of rotavirus SA11-infected MA104 cells with strictly defined reagents we produced monoclonal antibodies against synthetic peptides from both outer capsid proteins VP4 (aa residues 228–241: QNTRNIVPVSIVSR) and VP7 (aa residues 319–326: SAAFYYRV) of simian rotavirus SA11. Two of the selected monoclonal antibodies proved to be reactive with determinants of SA11-infected MA104 rhesus monkey kidney cells, with purified SA11 as well as with the particular peptides used for immunization. The anti-VP4 antibody had a demonstrable neutralizing titer of 200 (50% focus reduction) whereas the anti-VP7 MuMAb revealed no detectable neutralizing activity. In peptide-inhibition experiments, the corresponding peptide inhibited its MuMAb whereas the noncorresponding peptide had no effect on antibody binding to intracellular viral antigen. Localization of VP7 was preceded by VP4 as shown by immunofluorescence microscopy.     

Mechanism of entry of human rhinovirus 2 into HeLa cells

Internalized human rhinovirus 2 (HRV2) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20 degrees but not at 34 degrees the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20 degrees prior to addition of monensin permits virus synthesis to occur, depending on the time of preincubation.     

Integrin-mediated entry into S phase of human gastric adenocarcinoma cells

The integrins are a family of integral membrane receptors that participate in binding to various extracellular and cell surface proteins during adhesion, migration, and homing of normal and neoplastic cells. In this study, we characterized the involvement of integrins in mediating the growth of an adhesion-dependent gastric adenocarcinoma line, ST2. This line was distinguished and selected for study based on its inability to grow when suspended in soft agar or plated on poly(2-hydroxyethyl methacrylate)-coated dishes. ST2 cells arrested in G0/G1 of the cell cycle when deprived of adhesion to substrate. Using purified matrix components, collagen was found to be highly active in promoting 1 integrin-mediated cell attachment and spreading. Subsequent to spreading on collagen, the cells were released from G0/G1 block and progressed into S phase. Monoclonal antibodies to 2 or 1 integrin blocked the reinduction of both cell spreading and entry into S phase. These studies suggest that during the metastatic process, integrin receptor interaction with the insoluble matrix may be an important step leading to proliferation of some tumors.     

Human rotavirus infection enhances invasiveness of enterobacteria in MA-104 cells

To study the interaction between common pathogens causing infectious diarrhea in humans, MA 104 cell cultures were infected with human rotavirus and Salmonella typhimurium or Shigella flexneri or enteroinvasive Escherichia coli (EIEC) concomitantly. When MA-104 cells were preinfected with human rotavirus, invasiveness of S. typhimurium was significantly enhanced. The enhancement was evident after 48 h of virus preincubation. At this time virus specific antigens were demonstrated in the cell cultures. Also, the invasiveness of S. flexneri and EIEC was enhanced after virus preincubation, though not significantly before 72 h. When the virus preincubation time was prolonged to 96 h a further increase in invasive ability was observed. No cytopathogenic effect of the virus on the cells was demonstrated. Two control strains of non-enteropathogenic E. coli did not show any invasiveness in MA-104 cells pretreated with virus. The results indicate a specific interaction between rotavirus infected cells and facultatively intracellular enteropathogenic bacteria.     

Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes

Summary The entry modes of Japanese encephalitis (JE) and dengue-2 (DEN-2) viruses into C6/36 mosquito cells and of DEN-2 virus into human peripheral blood monocytes in vitro were studied. Inoculation of either JE or DEN-2 virions into C6/36 cells resulted in direct penetration of the virions into the cytoplasm at the cell surface in 3 stages. At stage 1, virions attached to the plasma membrane of host cells by their envelope spikes; at stage 2, the virion envelopes approximated to and eventually overlapped the host plasma membrane, and in the process the plasma membrane at the attachment sites dissolved; and, at stage 3, virions penetrated into the cytoplasm through the plasma-membrane disruptions created at the adsorption sites. Virions themselves apparently disintegrated at or near the penetration sites, for no virions were seen in the deeper cytoplasm. Coated pits did not form at the virion attachment sites, and virion-containing vesicles were not found in the cytoplasm. In the entry of DEN-2 virus into human peripheral blood monocytes, virions were found, adsorbed onto the external surface of the plasma membrane and attached to the luminal surface of macropinocytic vacuolar membranes. The latter apparently occurred as the result of ruffling and macropinocytic activities of the cells. At both sites virions penetrated into the cytoplasm through the plasma or vacuolar membrane in the same manner as they did through the plasma membrane of C6/36 cells. No evidence of viral entry by receptor-mediated endocytosis was observed. Implications of the entry mode of the mosquito cell-generated DEN-2 virus into human peripheral blood monocytes to an early process of natural, mosquito-transmitted infection is discussed.     

Pseudomonas aeruginosa selective adherence to and entry into human endothelial cells.

The pathogenesis of Pseudomonas aeruginosa disseminated infections depends on bacterial interaction with blood vessels. We have hypothesized that in order to traverse the endothelial barrier, bacteria would have to adhere to and damage endothelial cells. To test this hypothesis, we studied the adherence to human endothelial cells in primary culture of the piliated P. aeruginosa strain PAK and of two isogenic nonpiliated strains: PAK/p-, which carries a mutation in the pilin structural gene, and PAK-N1, a mutant defective in the regulatory rpoN gene. PAK adhered significantly more than did the pilus-lacking strains. P. aeruginosa was also taken up by endothelial cells, as determined by quantitative bacteriologic assays and by transmission electron microscopy. This internalization of P. aeruginosa seems to be a selective process, since the piliated strain was taken up significantly more than the nonpiliated bacteria and the avirulent Escherichia coli DH5 alpha, even following bacterial centrifugation onto the cell monolayers. A significant fraction of the internalized P. aeruginosa PAK was recovered in a viable form after 6 h of residence within endothelial cells. Progressive endothelial cell damage resulted from PAK intracellular harboring, as indicated by the release of lactate dehydrogenase. An increasing concentration of PAK cells was recovered from the extracellular medium with time, suggesting that ingested bacteria were released from endothelial cells and multiplied freely. We speculate that in vivo the ability of some P. aeruginosa strains to resist intracellular residence would afford protection from host defenses and antibiotics and that the release of viable bacteria into bloodstream may represent a central feature of the pathogenesis of bacteremia in compromised patients.     

Primaquine and lysosomotropic amines inhibit malaria sporozoite entry into human liver cells

The binding and entry of Plasmodium berghei sporozoites to human hepatoma HepG2 cells is inhibited in a dose-dependent manner by primaquine, chloroquine and other lysosomotropic amines. The site of action of these agents appears to be the hepatoma cell itself, not the sporozoite. While this inhibitory effect of primaquine is rapidly reversible, the precise mechanism responsible for this effect is not presently known.     

Ultrastructural study of Listeria monocytogenes entry into cultured human colonic epithelial cells.

Evidence that Listeria monocytogenes enters Caco-2 cells through the apical surface is presented. Attachment of bacteria to host cells seems to induce modifications of microvilli which are either in direct contact with the bacterial surface or in close vicinity, resulting in the formation of lamellipodia involved in the cellular uptake of the bacteria. Such modifications are not induced by L. monocytogenes SLCC 53, which carries a deletion in the prfA gene, although attachment of this mutant to Caco-2 cells occurs. Listeria innocua does not attach well to Caco-2 cells and also fails to cause structural alterations of the microvilli. Treatment of confluent monolayers of Caco-2 cells with ethylene glycol-bis(beta-aminoethyl ether)- N,N,N1,N1-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly reduced the uptake of Listeria cells. Attachment and invasion of L. monocytogenes was not accompanied by accumulation of filamentous actin around the entering bacterial cell.     

Rickettsia conorii entry into Vero cells.

The entry of rickettsiae into eukaryotic cells is mediated by an induced phagocytosis, but rickettsiae have never been observed in a closed phagocytic vacuole. In this study, Rickettsia conorii entry into Vero cells was observed by transmission electron microscopy during a period of 3 to 20 min after bacterium-cell contact. The entry occurred within 3 min after bacterium-cell contact, and R. conorii was observed in the process of engulfment, within a phagocytic vacuole, or free in the cytosol. Escape from the phagosome is a very rapid step since phagosome lysis was only occasionally observed. By 12 min, 90% of bacteria were internalized and half were free in the cytosol. This report confirms that rickettsiae penetrate nonphagocytic cells by induced phagocytosis and is the first demonstration of rickettsiae within a complete phagocytic vacuole.     

A comparative study of entry modes into C6/36 cells by Semliki Forest and Japanese encephalitis viruses

Summary The entry modes of Semliki Forest virus and Japanese encephalitis virus into C6/36 cells were compared by electron microscopic observation. At physiological pH, the two viruses showed characteristically different entry modes. Following attachment to the plasma membrane, many SF virions appeared within plasma membrane invaginations and cytoplasmic vesicles; on the other hand, JE virions remained to be found exclusively at the cell surface, with no virions appearing within cytoplasmic vesicles. Electron microscopic observation, therefore, indicated that SF virus entered C6/36 cells by receptormediated endocytosis, while JE virus penetrated the cells at the surface and disintegrated at or near the adsorption sites. At pH 5.8, SF virus also entered C6/36 cells by direct penetration at the cell surface. On the basis of the present and other findings, the following working hypotheses are presented for future investigations: (a) at physiological pH, the fusion protein of SF virus is in an inactive state and needs to be activated by acidic pH within the endosome in order to act on the host-cell membrane, but that of JE virus is in an active state and is capable of dissolving the host plasma membrane at the cell surface immediately after the attachment; (b) the states of viral fusion proteins (inactive or active) at the time of viral attachment to the cell surface determine which of the two entry modes these viruses follow.     

The entry of Junin virus into Vero cells

Summary The entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryoctye formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.