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.     

Membrane phospholipid asymmetry in Semliki Forest virus grown in BHK cells

The distribution of phospholipids across the membrane bilayer of Semliki Forest virus grown in BHK cells has been examined by treating the virus with bee venom phospholipase A₂ and sphingomyelinase C from Staphylococcus aureus. From the amounts of different phospholipids which are degraded rapidly (half-time about 1 min for phospholipase A₂) we calculate that in virus isolated 16 h after infection about 95% of sphingomyelin, 55% of phosphatidylcholine, 20% of phosphatidylethanolamine and less then 5% of phosphatidylserine is present on the outer leaflet of the virus envelope. Less than 5% of the virus was permeable to macromolecules before or after treatment with phospholipases as judged by accessibility of the genome to external ribonuclease. A much slower (half-time about 1 h) breakdown by phospholipase A₂ of originally inaccessible phosphatidylcholine and phosphatidylethanolamine appeared to be due to an enzyme-induced loss of lipid asymmetry since the original asymmetric distribution of phospholipids was maintained for several hours when the virus alone was incubated at 37°C. However, virus incubated for 20 h at 37°C showed a marked loss of phosphatidylethanolamine and phosphatidylserine asymmetry and a greater susceptibility to lysis by longer treatment with phospholipase A₂.     

Comparison of the Lipids of Intracellular and Extracellular Rabies Viruses

The lipid composition of both intracellular and extracellular forms of the ERA strain of rabies virus grown in BHK/21 cells was determined. The lipids from purified preparations of both intracellular and extracellular virus yielded 57 and 58% neutral lipid, respectively. The phospholipids of the intracellular and extracellular virus constituted 43 and 42%, respectively. Triglyceride and cholesterol appear to be the major neutral lipids, whereas sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine comprise the major bulk of phospholipid in both virus types. The molar ratio of cholesterol to phospholipid was 0.87 (intracellular) and 0.92 (extracellular). On the basis of the data presented, it is reasonable to assume that the lipids of both intracellular and extracellular rabies virus are similar.     

Defective interfering particles of Sindbis virus do not interfere with the homologous virus obtained from persistently infected BHK cells but do interfere with Semliki Forest virus.

Defective interfering particles derived from wild-type Sindbis virus no longer interfere with the infectious virus cloned from BHK cells persistently infected with Sindbis virus for 16 months. These particles do interfere with the replication of Semliki Forest virus.     

Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2.

About 50% of Semliki Forest virus-specific nonstructural protein nsP2 is associated with the nuclear fraction in virus-infected BHK cells. Transport into the nucleus must be specific, since only trace amounts of nsP3 and nsP4 and about 13% of nsP1, all derived from the same polyprotein, were found in the nucleus. Subfractionation of [35S]methionine-labeled Semliki Forest virus-infected cells showed that 80 to 90% of the nuclear nsP2 was associated with the nuclear matrix. Indirect immunofluorescence, with anti-nsP2 antiserum, showed the most intensive staining of structures which by Nomarski optics appeared to be nucleoli. In the presence of 1 to 5 micrograms of dactinomycin per ml the nuclei were stained evenly and no nucleoli could be found. Transport of nsP2 into the nucleus occurred early in infection and was fairly rapid. A cDNA encoding the complete nsP2 was isolated by the polymerase chain reaction technique and ligated into a simian virus 40 expression vector derivative. When BHK cells were transfected with this pSV-NS2 vector by the lipofection procedure, nsP2 was expressed in about 1 to 5% of the cells, as shown by indirect immunofluorescence. In positively transfected cells the immunofluorescence stain was most intensive in the nucleoli. Thus, Semliki Forest virus-specific nsP2 must have information which directs it into the nuclear matrix and, more specifically, into the nucleoli.     

Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus.

Three Aedes albopictus (mosquito) cell lines persistently infected with Sindbis virus excluded the replication of both homologous (various strains of Sindbis) and heterologous (Aura, Semliki Forest, and Ross River) alphaviruses. In contrast, an unrelated flavivirus, yellow fever virus, replicated equally well in uninfected and persistently infected cells of each line. Sindbis virus and Semliki Forest virus are among the most distantly related alphaviruses, and our results thus indicate that mosquito cells persistently infected with Sindbis virus are broadly able to exclude other alphaviruses but that exclusion is restricted to members of the alphavirus genus. Superinfection exclusion occurred to the same extent in three biologically distinct cell clones, indicating that the expression of superinfection exclusion is conserved among A. albopictus cell types. Superinfection of persistently infected C7-10 cells, which show a severe cytopathic effect during primary Sindbis virus infection, by homologous virus does not produce cytopathology, consistent with the idea that cytopathology requires significant levels of viral replication. A possible model for the molecular basis of superinfection exclusion, which suggests a central role for the alphavirus trans-acting protease that processes the nonstructural proteins, is discussed in light of these results.     

Quantitation of Semlike Forest virus RNAs in infected cells using 32-P equilibrium labelling.

In vitro cultured BHK and HeLa cells were labelled for several cell division cycles with 32-P-phosphate until they were equilibrated with radiophosphorus. After infection with Semliki forest virus (or mock-infection) these cells were analyzed for viral and ribosomal RNA by sucrose gradient centrifugation. From their radioactivities the mass of each RNA species was calculated. It was found that the BHK and HeLa cells contained on average 11.0 plus or minus 3.1 pg and 6.3 plus or minus 1.9 pg of ribosomal RNA (28 S + 18 S) respectively per cell. At the end of the viral growth cycle, i.e. at 8 h post infection the average mass of viral genome produced per cell was 1.0 -1.9 pg and 0.3 - 0.5 pg in BHK and HeLa cells respectively, of which only 1/10 to 1/20 was released as mature virus particles. The amount of the second major virus specific messenger, the 26 S RNA, was estimated from its ratio to the viral genome after labelling with 3-H-uridine in the presence of actinomycin D. These two viral RNAs were found to be present in roughly equimolar amounts.     

Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.     

Protein synthesis in cells infected with Semliki Forest virus is not controlled by intracellular cation changes

Treatment of BHK cells with 1 microM nigericin results in a 55% decrease in K+ and a 3.3-fold increase in intracellular Na+; protein synthesis under these conditions is depressed by 35%. In BHK cells infected with Semliki Forest virus (SFV), protein synthesis is depressed by 76% 6.5 h after infection; intracellular K+ is unchanged, and intracellular Na+ is increased 1.8-fold at this time. These results suggest that the increase in intracellular Na+ in SFV-infected BHK cells does not adequately account for the decrease in protein synthesis, and makes it likely that an increased Na+ concentration is a consequence, not a cause, of alterations in protein synthesis in virally-infected cells. No evidence was obtained for the purported [Alonso, M. A. and Carrasco, L. (1980) Eur. J. Biochem. 109, 535-540; (1981) Eur. J. Biochem. 118, 289-294; (1981) FEBS Lett. 127, 112-114] ability of 1 microM nigericin to permeabilize' cells.     

Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells.

We investigated the biogenesis of type I cytopathic vacuoles (CPVIs) in Semliki Forest virus (SFV)-infected cells by immunofluorescence and electron microscopy. By using the ts1 mutant of SFV at the restrictive temperature to avoid superinfection, we showed that the multiplicity of infection affects the time of appearance but not the number of CPVIs in a cell. Formation of CPVIs did not require incoming virus particles, because they were found in BHK cells transfected with infectious RNA from the SFV prototype strain or ts1 mutant. When the SFV gene for nsP3 was expressed alone in BHK cells, the nsP3 protein was localized to numerous vesiclelike structures and large vacuoles. The nsP3 protein may function as an anchoring protein for the RNA replication complex of SFV.     

Characterization of a small, nonstructural viral polypeptide present late during infection of BHK cells by Semliki Forest virus.

BHK cells, late in infection with Semliki Forest virus, were found to contain a small virus-specific polypeptide not found in the mature virion. This polypeptide had an apparent molecular weight of 6,000 and is referred to here as the 6K protein. No [2-3H]mannose was incorporated into 6K, and hence it does not appear to be a glycoprotein. This protein appears to be a primary translation product of the subgenomic 26S mRNA, which encodes the viral structural proteins. The genes encoding the viral structural proteins are arranged on the message in the order of 5'-C-E3-E2-E1-3'. We have found that the gene coding for 6K is located to the 3' side of the gene encoding E2. Subcellular fractionation of pulse-labeled cells infected with Semliki Forest virus demonstrated that 6K, like the viral glycoproteins p62 and E1, was present predominantly in the rough microsomal membrane fraction. 6K appears to be analogous, therefore, to the nonstructural 4.2K protein present in cells infected with Sindbis virus.     

Antiviral activity released from Aedes albopictus cells persistently infected with Semliki forest virus.

Aedes albopictus (mosquito) cells persistently infected with Semliki Forest virus released an agent which inhibited virus production by A. albopictus cells infected with homologous virus. Inhibition of virus production was accompanied by a marked reduction in the synthesis of viral RNA and viral proteins. Expression of the antiviral effect was prevented by pretreatment of cells with actinomycin. No analogous antiviral activity was detected in culture fluids of A. albopictus cells persistently infected with a flavivirus (Kunjin virus) or a bunyavirus (Bunyamwera virus).     

Generation of defective interfering particles of Semliki Forest virus in a clone of Aedes albopictus (mosquito) cells.

Serial undiluted passage of Semliki Forest virus in a clone of Aedes albopictus cells resulted in a marked decrease in infectious virus yields due to the generation and accumulation of defective interfering particles. Virus from the third passage had a high particle/infectivity ratio and interfered specifically with homologous but not heterologous standard virus replication. Two RNA species of molecular weights 0.78 X 10(6) and 0.61 X 10(6) were the major RNA components of purified passage 4 virus. These RNA species were also the predominant virus RNA species detected in cells infected with passage 3 virus. Synthesis of standard virus RNA and virus-specified protein was much reduced in passage 3 virus-infected cells. Interference with standard virus replication and the synthesis of large amounts of defective interfering RNA were also observed in chicken embryo cells infected with passage 3 virus from mosquito cells.     

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.     

Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1.

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.     

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.     

Different membrane anchors allow the Semliki Forest virus spike subunit E2 to reach the cell surface.

The Semliki Forest virus spike subunit E2, a membrane-spanning protein, was transported to the plasma membrane in BHK cells after its carboxy terminus, including the intramembranous and cytoplasmic portions, was replaced by respective fragments of either the vesicular stomatitis virus glycoprotein or the fowl plague virus hemagglutinin. The hybrid proteins were constructed by cDNA fusion. Upon a transient expression they could be localized at the cell surface by immunofluorescence with specific antibodies directed against any of the protein fragments.     

The 37-kDa/67-kDa laminin receptor acts as the cell-surface receptor for the cellular prion protein.

Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.     

Structure and dynamics of the phosphatidylcholine and the phosphatidylethanolamine head group in L-M fibroblasts as studied by deuterium nuclear magnetic resonance.

Mouse fibroblast L-M cells were grown in tissue culture medium containing selectively deuterated choline or ethanolamine. Both compounds were incorporated into the corresponding phospholipids at levels greater than 50% thus leading to a selective deuteration of these phospholipid head groups. Choline and ethanolamine were labeled at either the alpha- or the beta-carbon atom and well-resolved deuterium and phosphorus n.m.r. spectra were obtained from intact cells, crude plasma membranes and lipid extracts, leading to the following conclusions. (i) A large fraction, if not all, of the phospholipids in the intact L-M cell membranes were organized in a liquid crystalline bilayer. (ii) The phosphoethanolamine and the phosphocholine head group conformation were found to be remarkably similar in pure lipid bilayers and in intact L-M cell membranes with the head group dipoles being oriented parallel to the membrane surface. (iii) The deuterium T1 spin lattice relaxation times fell in the range of 7-25 ms and were similar in intact L-M cells and in pure lipid model membranes, suggesting that the two head groups are not involved in strong interactions with membrane proteins. The rotational diffusion rate of the two head groups was reduced by at least a factor of 10 compared to molecules of the same size in aqueous solution. (iv) The phosphocholine head group was sensitive to the size and sign of membrane surface charges as verified in mixing experiments with charged lipids. In L-M cell membranes the phosphocholine appeared to sense an electrically neutral environment in spite of the fact that L-M cell membranes contain 10-20% negatively charged lipids.     

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.