Virus accumulation by the rock oyster Crassostrea glomerata.

The accumulation of virus by the New Zealand rock oyster Crassostrea glomerata has been studied in a static seawater system using radioactively labeled reovirus type III and Semliki Forest virus. The uptake of virus was found to be less rapid than for the bacterium Escherichia coli and to be unaffected by the presence of the marine alga Dunaliella primolecta in the seawater. Accumulation was dependent on virus concentration, with saturation achieved at 4 X 10(10) reovirus particles per oyster, implying that an oyster possesses a large but finite number of sites for virus adsorption. When the rates of uptake of two viruses of similar size but differing surface properties were compared, the rate of accumulation of the lipoprotein-enveloped Semliki Forest virus was found to be less than that for the protein-enclosed reovirus. This observation, together with the finding that the oyster shell has a strong affinity for virus, suggests that surface properties, rather than size, are the principal factors governing the accumulation of viruses by filter-feeding marine bivalves.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

Influence of the infection with lipid-containing viruses on the metabolism and pools of phospholipid precursors in animal cells

The influence of infection with three different lipid-containing RNA viruses, Newcastle disease virus, fowl plague virus, and Semliki Forest virus on the phosphatidylcholine precursors of chick embryo cells and of baby hamster kidney (BHK) cells has been measured. In chick embryo cells infection with Newcastle disease virus does not influence the energy charge, or the distribution and absolute pool sizes of the precursors or the choline phosphotransferase activity. In chick embryo cells infected with fowl plague virus the CDP-choline pool increases because of an inhibition of the choline phosphotransferase activity. The phosphorylcholine and CTP pools are smaller in infected cells when compared with mock-infected ones, although the energy charge is not influenced by infection. In chick embryo cells as well as in BHK cells the energy charge is diminished by infection with Semliki Forest virus. Therefore the CTP and phosphorylcholine pools are decreased. The CDP-choline pool in chick embryo cells becomes extremely small after infection with Semliki Forest virus because of a significant stimulation of the choline phosphotransferase. In BHK cells infected with Semliki Forest virus the opposite effect is observed. There are also severe effects on the uptake of the labeled precursors by infection. One and the same virus (Semliki Forest virus) has two completely different effects on the phosphatidylcholine precursors when infecting two different cell types. If one and the same cell type (chick embryo cells) is infected with three different lipid-containing RNA viruses also completely different effects on the phosphatidylcholine precursors were observed. Thus, each virus develops its own strategy to influence the lipid metabolism of the host cell, depending also on the choice of the host. This explains the many disturbing contradictory results described in the literature about the influence of lipid-containing viruses on the lipid metabolism of the host.     

Analysis of alphavirus polypeptides by two dimensional polyacrylamide gel electrophoresis

Semliki Forest, Sindbis and Chikungunya viruses were grown and radio-labeled with [³H]-amino acids in Vero cells. Analysis of virus infected cell lysates by two dimensional polyacrylamide gel electrophoresis resulted in detection of polypeptides of molecular, weights corresponding to those of E1, P62, ns60, ns70/72 for Semliki Forest virus, the C, E1, 6K, 14K, PE2, P97, ns60, ns82 for Sindbis virus and E1. P62, P97, ns70/72 for Chikungunya virus. Charge and molecular weight heterogeneity in the precursor polypeptide P62 of Semliki Forest virus was detected. Structural poly-peptides e.g. E1 and E2 of Semliki Forest virus and C, E1, E2 of Sindbis virus and E1 of Chikungunya virus were detected when purified radiolabeled virus preparations were analyzed by two dimensional polyacrylamide gel-electrophoresis. Membrane glycoprotein E1 and E2 of Semliki Forest and E1 of Sindbis and Chikungunya viruses exhibited charge heterogeneity. In contrast to the marked difference in isoelectric points of E1 and E2 of Sindbis virus; E1 and E2 of Semliki Forest virus had almost identical isoelectric points.     

Do cells treated with human interferon survive virus infection?

Abstract HeLa cells pretreated with human lympho-blastoid interferon (Hu IFN-α (Ly)), at concentrations up to 100 IU / ml and infected with moderate multiplicities of encephalomyocarditis (EMC) virus (10 PFU / cell) died 1 or 2 days after infection. However, if cells were repeatedly treated with high doses of IFN (800 IU / ml) they survived infection by EMC virus for at least a month. Cells survived Semliki Forest virus (SFV) infection when even lower IFN concentrations were used. By contrast infection of IFN-treated HeLa cells with other RNA-containing viruses, such as poliovirus, vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and reovirus type 3 resulted in cell death. Similarly, infection with a number of DNA-containing viruses such as adenovirus type 5, Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and vaccinia virus killed cells. The results are discussed in the light of different models for the molecular mechanism of action of interferon.     

Inactivation of Viruses by Benzalkonium Chloride

Benzalkonium chloride (as Roccal or Zephiran) was found to inactivate influenza, measles, canine distemper, rabies, fowl laryngotracheitis, vaccinia, Semliki Forest, feline pneumonitis, meningopneumonitis, and herpes simplex viruses after 10 min of exposure at 30 C or at room temperature. Poliovirus and encephalomyocarditis virus were not inactivated under the same conditions. It was concluded that all viruses tested were sensitive except members of the picorna group. The literature was reviewed.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes.

Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.     

Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus.

The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant.     

Pathogenesis of in utero infections with abortogenic and non-abortogenic alphaviruses in mice.

The initial stages of infection of pregnant mice at gestation day 11 with either the T48 strain of Ross River virus or avirulent Semliki Forest virus are similar. With both infections, a hematogenous spread of virus to the placenta occurs. The viruses subsequently replicate to high titer in all placentas and are able to persist in the presence of specific maternal antiviral antibodies. There is a delay of at least 1 to 2 days between the initial detection of virus in the placenta and the onset of fetal infection. With Semliki Forest virus, abortion occurred in all mothers and appeared to be preceded by infection of all fetuses. However, when Semliki Forest virus was given at other stages of pregnancy, abortion was less common, and in all non-aborted pregnancies at least one uninfected fetus was observed. This situation was similar to that with Ross River virus, in which abortion was not observed and fetal infection and death were only seen in a proportion of fetuses. Within each pregnancy, the outcome of the two in utero infections appeared to result from similar mechanisms, with the fate of an individual fetus depending upon the timing of the passive transfer of anti-viral immunoglobulin G from the mother relative to the timing of fetal infection by virus from the placenta. Although the passive maternal immunoglobulin G protected susceptible fetuses against infection, antibody did not mediate in utero recovery of infected fetuses or clear placental infection.     

A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses

Successful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it reduced viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a previously unappreciated role for WDR81 in promoting the replication of viruses that transit through late endosomes.     

Uptake and survival of enteric viruses in the blue crab, Callinectes sapidus.

Uptake of poliovirus 1 by the blue crab, Callinectes sapidus, was measured to assess the likelihood of contamination by human enteric viruses. Virus was found in all parts of the crab within 2 h after the crab was placed in contaminated artificial seawater. The highest concentrations of virus were found in the hemolymph and digestive tract, but the meat also contained virus. The concentration of virus in the crabs was generally less than in the surrounding water. Changes in salinity did not substantially affect the rate of accumulation. An increase in temperature from 15 to 25 degrees C increased the rates of both uptake and removal. Poliovirus survived up to 6 days in crabs at a temperature of 15 degrees C and a salinity of 10 g/kg. When contaminated crabs were boiled, 99.9% of poliovirus 1, simian rotavirus SA11, and a natural isolate of echovirus 1 were inactivated within 8 min. These data demonstrate that viruses in crabs should not pose a serious health hazard if recommended cooking procedures are used.     

Characterization of a common high-affinity receptor for reovirus serotypes 1 and 3 on endothelial cells.

During viremia, viruses may be cleared from the bloodstream and taken up by specific organs. The uptake of virus from the bloodstream is dependent on the association of viral particles with endothelial cells that line the luminal surfaces of large and small blood vessels. To understand the nature of this interaction, we have studied the binding of reovirus serotypes 1 and 3 to these cells in vitro. Both serotypes of reovirus productively infected endothelial cells. By using [35S]methionine-biolabeled reovirus as a tracer ligand, we found that both viruses rapidly bind to endothelial cells and that equilibrium is reached after 4 h. The binding of the radiolabeled viruses was saturable and mediated by a homogeneous population of cellular receptors with very high affinity (Kd = 0.5 nM) for the virus ligands. Both serotypes bind to the same receptor, since the attachment of each radiolabeled serotype is inhibited by both the homologous and heterologous unlabeled virus. Exposure of labeled virus to monoclonal antibodies directed against the viral hemagglutinin (sigma 1 protein) inhibited binding, demonstrating that the attachment of reovirus to endothelial cells is mediated by the hemagglutinin for both serotypes. By using a novel ligand-blotting assay, the binding of both viruses to a 54,000-dalton protein could be demonstrated. The binding of each radiolabeled serotype to this protein was inhibited by the homologous and heterologous unlabeled serotype. By using cell fractionation after homogenization, we demonstrated that this 54-kilodalton protein is a membrane protein, in agreement with its proposed role as a cell surface receptor for reovirus serotypes 1 and 3.     

Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus.

The efficiencies with which homologous and heterologous proteins are incorporated into the envelope of Moloney murine leukemia virus (M-MuLV) have been analyzed by utilizing a heterologous, Semliki Forest virus-driven M-MuLV assembly system and quantitative pulse-chase assays. Homologous M-MuLV spike protein was found to be efficiently incorporated into extracellular virus particles when expressed at a relatively low density at the plasma membrane. In contrast, efficient incorporation of heterologous proteins (the spike complex of Semliki Forest virus and a cytoplasmically truncated mutant of the human transferrin receptor) was observed only when these proteins were expressed at high densities at the cell surface. These results imply that homologous and heterologous proteins are incorporated into the M-MuLV envelope via two distinct pathways.     

Identification of a novel function of the alphavirus capping apparatus. RNA 5'-triphosphatase activity of Nsp2

Both genomic and subgenomic RNAs of the Alphavirus have m(7)G(5')ppp(5')N (cap0 structure) at their 5' end. Previously it has been shown that Alphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5' gamma-phosphate of the nascent viral RNA chain is removed by a specific RNA 5'-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5' end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.     

Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells.

Hyperimmune, but not normal immune, monospecific antiserum made to capsid protein of Sindbis virus (SIN) was found to cause cytolysis equally well of both SIN- and Semliki Forest virus-infected L929 cells in antibody-dependent, complement-mediated cytotoxicity assays. The cell surface reactivity of the hyperimmune antiserum was also demonstrated by solid-phase radioimmune assays with unfixed infected cells or infected cells fixed with low concentrations of glutaraldehyde (0.025%) before reactivity with antisera. Higher concentrations of glutaraldehyde lowered the sensitivity of detection. Purified SIN capsid protein specifically inhibited antibody-dependent, complement-mediated cytotoxicity by the monospecific anti-capsid protein serum on SIN- and Semliki Forest virus-infected target cells. That hyperimmune anti-SIN serum also cross-reacts with capsid protein on the surface of Semliki Forest virus-infected cells was suggested by the fact that capsid protein inhibited cross-cytolysis in the antibody-dependent, complement-mediated cytotoxicity assay. The latter antiserum was collected after repeated injections of purified virions over a 9-month period. The results suggest that hyperimmune monospecific antisera made to SIN capsid protein or hyperimmune antisera to SIN or Semliki Forest virions detect homologous and cross-reacting capsid protein determinants on the surface of infected cells.     

Metastatic tumor cell variants with increased resistance to infection by Semliki Forest virus

Semliki Forest virus (SFV) is an interesting virus for cell interaction studies because it binds directly to the cells' major histocompatibility antigens. We used this reagent to study the expression and functional properties of H-2 molecules on murine tumor lines that are closely related but differ greatly in metastatic capacity. Tumor cell variants with high metastatic capacity showed an increased resistance to virus infection, an effect that was selective for SFV. Although the high metastatic tumor lines did not express less H-2 antigens than the low metastatic ones, they bound much less of the SFV viral glycoproteins.     

Effects of monovalent cations on Semliki Forest virus entry into BHK-21 cells

Infection of mammalian cells with Semliki Forest virus requires the endocytosis of the virus, its delivery to prelysosomal endosomes, and fusion of the viral envelope with the endosome membrane. Previous studies have indicated that the low endosomal pH triggers a conformational change in the viral spike glycoproteins rendering them fusogenic. In this paper, we demonstrate an additional factor(s) which regulates virus fusion in endosomes. We found that Semliki Forest virus is unable to penetrate or infect baby hamster kidney (BHK-21) cells grown in medium containing reduced Na+ concentrations. Virus endocytosis and degradation are nearly normal, the virus is transported to endosomes where a characteristic low pH-induced loss of trypsin-sensitivity of the E1 spike glycoprotein occurs. Nevertheless, the viral envelope fails to fuse with the endosomal membrane and the viral RNA is not released into the cytosol. As judged by the uptake of the voltage-sensitive probe [3H]triphenylmethyl phosphonium we observed a close correlation between conditions which inhibit virus infection and which cause depolarization of the cells. We propose that in intact cells, the fusion of Semliki Forest virus with the endosome membrane depends not only on acidic endosomal pH, but also on the maintenance of the potential.     

A mathematical model of the trafficking of acid-dependent enveloped viruses: application to the binding, uptake, and nuclear accumulation of baculovirus

A quantitative understanding of virus trafficking would be useful in treating viral-mediated diseases, developing protocols for viral gene therapy, designing infection regimens for viral expression systems, and optimizing vaccine and recombinant protein production. Here, we present a mathematical model of the attachment, internalization, endosomal fusion, lysosomal routing, and nuclear accumulation of baculovirus in SF21 insect cells. The model accounts for multivalent bond formation of the virus with cell surface receptors. The model mimics accurately the experimental trafficking dynamics of the virus at both low and high virion to cell ratios, and estimates a receptor number of 11,000 per cell. A significant amount of virus was degraded intracellularly. Independent of the virion to cell ratio, half of the internalized virus was degraded with the rest accumulating in the nucleus. The formalism used in the model may be generally useful for other acid-dependent enveloped viruses. A subset of the model has been used previously to describe the trafficking of Semliki Forest virus, an acid-dependent enveloped RNA virus.Two pathways have previously been implicated for the in vitro entry of the budded form of the baculovirus: adsorptive endocytosis and plasma membrane fusion. Experimental evidence is presented which strongly suggests that the physical number of viruses entering by plasma membrane fusion is not significant relative to receptor-mediated endocytosis. (c) 1997 John Wiley & Sons, Inc., Biotechnol Bioeng 54: 468-490, 1997.     

Infectious particles derived from Semliki Forest virus vectors encoding murine leukemia virus envelopes.

Semliki Forest virus vectors encoding murine leukemia virus (MLV) envelope protein with a truncated cytoplasmic tail generate submicrometer, cell-associated, membranous particles that transmit replication-competent vector RNA specifically to cells bearing the MLV receptor. Such "minimal" viruses could have applications as retroviral vaccines or in the study of virus evolution.