Solubilized RNA replication complex from Semliki Forest virus-infected cells

The postmitochondrial pellet from SFV-infected cells was incubated under in vitro RNA-synthesizing conditions in the presence of [³H]UTP to label nascent chains in the RNA replication complex. The solubilized complex was purified by centrifugation in a glycerol gradient followed by a sucrose gradient in which it sedimented with a peak at 34 S. It contained double-stranded RNA which by mild ribonuclease treatment was converted to replicative forms I, II, and III, indicating that it was derived from replicative intermediates synthesizing 42 S and 26 S RNA. A similar structure was also isolated from infected cells labeled with [³H]uridine early in infection. The only virus-specific proteins associated with the complex were ns70, ns72, and ns86. The presence of ns70 and ns72 was confirmed by tryptic peptide mapping.     

Messenger ribonucleoprotein complexes containingin vitro-synthesized 26S and 42S Semliki Forest virus RNA

Summary An extract derived from Semliki Forst virus (SFV) infected cells is described which catalyzes the synthesis of virus-specific RNAs. The newly-synthesized 26S and 42S RNAs are found complexed with protein as messenger ribonucleoproteins (mRNPs). These mRNPs either are non-membrane bound or are associated with large cytoplasmic lipoprotein membranes, and they are found as free mRNPs as well as mRNPs bound to ribosomal subunits, ribosomes, and polysomes. Following treatment with Tween 40 and deoxycholate, membrane-bound mRNPs containingin vitro-synthesized 26S RNA are dissociated and sediment at 33S. These membrane-dissociated mRNPs contain relatively little protein. In contrast, the free or ribosome-bound mRNPs, which are isolated as 30S to 160S particles, remain heterogeneous after detergent treatment and have a much higher protein content. Addition of purified, native 40S ribosomal subunits to the extract leads to the formation of complexes between the added ribosomal subunits and the newly-synthesized viral mRNA. Thein vitro-synthesized 26S and 42S RNAs participate in the assembly of translational initiation and elongation complexes.With 9 Figures     

Prevention of death in Semliki Forest virus-infected mice by administration of defective-interfering Semliki Forest virus

Adult mice inoculated with Semliki Forest virus (SFV) were protected from a lethal infection of the central nervous system by intranasal administration of defective-interfering (DI) SFV. DI SFV was prepared by eight passages at high m.o.i. in BHK 21 cells. Mice were treated with unpurified, unconcentrated tissue culture fluid which had been u.v.-irradiated to inactivate the infective virus present. Prevention of death was maximal when the DI virus was administered simultaneously with the infecting inoculum, and under the same conditions multiplication of infective virus in the brains of treated mice was reduced by 10(5)-fold. It was shown that DI SFV was propagated in mouse brains followed intranasal inoculation and it was concluded that protection was brought about through the intrinsic interfering capacity of the DI virus.     

Role of interferon-gamma in T-cell responses to Semliki Forest virus-infected murine brain cells

Primary brain cell cultures prepared from newborn C3H mice were infected with Semliki Forest virus (SFV) or treated with a beta-propiolactone-inactivated preparation of SFV (BPL-SFV). The effects of recombinant interferon-gamma (IFN-gamma) treatment on SFV replication, SFV antigen display, major histocompatibility complex (MHC) class I and class II antigen expression, susceptibility to lysis by SFV-specific cytotoxic T lymphocytes (CTL) and the ability to stimulate SFV-specific T lymphocytes to release IFN-gamma were determined. The IFN-gamma treatment prevented replication of SFV, as determined by incorporation of [3H]uridine into SFV-RNA, and reduced expression of SFV antigens on the cell surface, as determined by lysis with antibody and complement or indirect immunofluorescence. BPL-SFV-treated brain cells expressed no SFV antigen detectable by lysis with antibody and complement or indirect immunofluorescence. IFN-gamma increased expression of MHC class I and class II antigens, measured by indirect immunofluorescence, susceptibility to killing by alloreactive T-cell lines and ability to stimulate an allogeneic mixed lymphocyte reaction (MLR). Brain cells infected with SFV or treated with BPL-SFV were susceptible to killing by the CTL. The killing was MHC restricted and neither uninfected nor untreated cells were killed. IFN-gamma treatment prior to SFV infection or BPL-SFV treatment resulted in an augmentation of lysis by the CTL, indicating that even where SFV antigen expression is reduced or present at very low levels, in the context of enhanced MHC class I expression cells remain susceptible to CTL killing. Brain cells treated with BPL-SFV stimulated SFV-specific T cells to release IFN-gamma. Pretreatment of brain cells with IFN-alpha beta or IFN-gamma prior to BPL-SFV treatment markedly increased the ability of the cells to stimulate the SFV-specific T cells to release IFN-gamma. Release of IFN-gamma was MHC restricted and brain cells untreated with BPL-SFV did not stimulate IFN-gamma release. IFN-gamma released by T cells stimulated with BPL-SFV-treated brain cells increased class II MHC expression by brain cells as assessed by indirect immunofluorescence.     

In vitro translation of 42 S virus-specific RNA from cells infected with the flavivirus West Nile virus.

Virus-specific proteins synthesized in BHK cells infected with the flavivirus West Nile (WN) virus and in vitro using the virus-specific infectious 42 S plus-strand RNA as messenger RNA, have been studied. Mapping of the tryptic peptides indicates that the viral core protein V2 and the viral glycoprotein V3 do not share common sequences. No tryptic peptides identifiable by mapping were obtained from the viral membrane-associated protein V1. Seven apparently virus-specific intracellular proteins were detected by comparative SDS-PAGE of mock-infected and virus-infected [³⁵S]methionine-labeled cell lysates: p_i 15, p_i 20, p_i 27, p_i 37, p_i 49, p_i 71, and p_i 100 (the index i indicates the intracellular origin of the proteins, the number gives the apparent molecular weight in 10³ daltons). The proteins p_i 15 and p_i 49 represent the intracellular forms of the viral structural proteins V2 and V3, respectively. p_i 15 and V2 are not identical but differ slightly from each other. V1 has not been detected in infected cells. Mapping has shown that the other intracellular proteins (with the possible exception of p_i 37, which has not been analyzed) are unrelated to either V2 or V3 and do not share common sequences. They represent nonstructural proteins. The total molecular weight of the apparently unrelated nonstructural and structural proteins is about 290,000 daltons. Data obtained in a number of laboratories have shown that a virus-specific 42 S RNA molecule, which is structurally indistinguishable from the viral genome, probably functions as mRNA for all virus-specific proteins in vivo. This RNA has been isolated from WN virus-infected cells and translated in vitro in the wheat germ and the rabbit reticulocyte lysate system. Digestion of the total [³⁵S]methionine-labeled proteins synthesized in vitro in either of both systems with trypsin followed by peptide mapping has shown that the great majority of the resulting peptides are present in the structural proteins V2 and V3 and vice versa. No evidence was obtained for the in vitro synthesis of nonstructural proteins. Proteins synthesized in the reticulocyte lysate were fractionated by SDS-PAGE and isolated polypeptides studied by peptide mapping. Polypeptides of molecular weights between 11,500 and 90,000 daltons were obtained. Their peptide maps indicate that all polypeptides are translated from a single initiation sequence. The map of the smallest in vitro synthesized polypeptide P_retic 11.5, having a molecular weight of 11,500 daltons, was almost identical to that of V2. The map of the largest protein synthesized in significant amounts in vitro, the protein P_retic 90, was very similar to the map of a mixture of the viral proteins V2 and V3. The analyses suggest the following gene order on the 42 S RNA: 5′-terminus-V2-V3-(V1, p_i 20, p_i 27, p_i 37, p_i 71, p_i 100)-3′-terminus. The order of genes indicated in brackets remains to be determined. Some implications of these results concerning the possible mode of translation of flavivirus-specific 42 S RNA are discussed.     

Novel vectors expressing anti-apoptotic protein Bcl-2 to study cell death in Semliki Forest virus-infected cells

Semliki Forest virus (SFV, Alphavirus) induce rapid shut down of host cell protein synthesis and apoptotic death of infected vertebrate cells. Data on alphavirus-induced apoptosis are controversial. In this study, the anti-apoptotic bcl-2 gene was placed under the control of duplicated subgenomic promoter or different internal ribosome entry sites (IRES) and expressed using a novel bicistronic SFV vector. The use of IRES containing vectors resulted in high-level Bcl-2 synthesis during the early stages of infection. Nevertheless, in infected BHK-21 cells translational shutdown was almost complete by 6h post-infection, which was similar to infection with appropriate control vectors. These results indicate that very early and high-level bcl-2 expression did not have a protective effect against SFV induced shutdown of host cell translation. No apoptotic cells were detected at those time points for any SFV vectors. Furthermore, Bcl-2 expression did not protect BHK-21 or AT3-neo cells at later time points, and infection of BHK-21 or AT3-neo cells with SFV replicon vectors or with wild-type SFV4 did not lead to release of cytochrome c from mitochondria. Taken together, our data suggest that SFV induced death in BHK-21 or AT3-neo cells is not triggered by the intrinsic pathway of apoptosis.     

Comparative studies on polyribosomal, nonpolyribosome-associated and viral 42 S RNA from BHK 21 cells infected with Semliki Forest virus.

Virus-specific RNA sedimenting at about 42 S on sucrose density gradients can be isolated from polyribosomes of BHK-21 cells infected with Semliki Forest virus and from intracellular nonpolyribosome-associated ribonucleoprotein particles sedimenting at about 55 S. These RNA species have been compared to the 42 S RNA of Semliki Forest virus particles by three different techniques: Infectivity, sedimentation on sucrose density gradients in formamide, and translation into virus-specific protein in vitro. The results of these experiments have shown that all three RNA species consist of intact, unbroken molecules, that they are equally infectious and that they contain the mRNA sequences for the viral core protein (the structural protein associated with the viral RNA in the virus particle). Some implications of these results concerning the possible utilization of the different 42 S RNA species for assembly of virus particles and the translation of the polyribosome-associated 42 S RNA in vivo are discussed.     

Conformational changes at pH 6 on the cell surface of Semliki Forest virus-infected Aedes albopictus cells

The mechanism of Semliki Forest virus-induced fusion from within at low pH was studied in Aedes albopictus cells. The fusion was found to occur in at least two steps, namely, a fast initial step which is pH dependent and temperature independent, and a second slower process which is pH independent and temperature dependent. The initiation step induced by low pH exposure constitutes an irreversible conformational change of a protein probably of viral origin located at the cell surface.     

Isolation and characterization of double stranded RNA containing infectious viral genome RNA from cells infected with Semliki Forest virus

Summary A double stranded virus specific RNA sedimenting at about 19S on sucrose density gradients has been isolated from BHK-21 cells infected with Semliki Forest virus (SFV). The molecule consists of double stranded RNA (ds RNA) since it is labeled with³H-uridine, is souble in 2m LiCl, resistant against treatment with DNase and RNase at 2×SSC, hydrolyzed by alkali treatment, has a sharp thermal melting point at 89° in 1/10 SSC, and an extended appearance under non denaturing conditions in the electronmicroscope. The following findings show that it consists of intact, infectious 42S RNA similar or identical to the genome RNA of SFV complexed to a complementary 42S minus strand RNA: 1. Denaturation converts the ds RNA into molecules cosedimenting with 42S RNA isolated from SFV particles. 2. About 50 per cent of the radioactivity of³H-uridine labeled 42S RNA molecules generated from 19S ds RNA by denaturation hybridizes to 42S viral RNA. 3. The specific infectivity of denatured 19S ds RNA is about half of that of similarly treated viral 42S RNA. Further properties of this molecule are discussed.With 5 Figures     

Sedimentational pattern of virus-specific RNA synthesized in Newcastle disease virus-infected cells treated with amino acid analogues

Summary The synthesis of virus-specific RNA in Newcastle disease virus-infected cells is strongly suppressed by amino acid analogues (p-fluorophenylalanine, ethionine and canavanine) if they are present throughout the reproduction cycle or added 2–4 hours p.i. The analogues exert only a partial inhibitory effect when added 6 hours p.i. Virus-specific RNA synthesized in cells exposed to ethionine or canavanine has essentially the same sedimentational pattern as RNA formed in the absence of the analogues, whereas p-fluorophenylalanine preferentially inhibits 35S RNA accumulation. The content of minus strands in different RNA classes is not changed in p-fluorophenylalanine-treated cells. 50S RNA formation, although readily suppressed by cycloheximide was not inhibited by the amino acid analogues tested.     

Cytoplasmic and nuclear virus-specific proteins in influenza virus-infected MDCK cells

Four virus-specific proteins were identified in nuclei purified from influenza virusinfected cells: the nucleocapsid protein (NP); two nonstructural proteins of 24,500 daltons (NS₁) and 11,000 daltons (NS₂) molecular weight; and the largest virion protein, protein P. The nucleocapsid protein was found to be distributed approximately equally between the nucleoplasmic and nucleolar fractions, whereas most of the other virus-specific proteins were associated with the nucleolar fraction. In the nucleoplasm, most, if not all, of the nucleocapsid protein was in the form of viral ribonucleoproteins.In addition to being in the nucleus, NS₁ has been found concentrated in cytoplasmic fractions containing polyribosomes and ribosomes (Pons, 1972; Compans, 1973). This suggested that NS₁ might be associated with ribosomes as a structural ribosomal protein and that, therefore, NS₁ was in the nucleolus for the purpose of being incorporated into newly assembled ribosomal subunits. Our studies show that in cytoplasmic extracts of infected cells, NS₁ was actually associated with ribosomes, but not as a structural protein; it was simply adsorbed to both the large and the small ribosomal subunits by salt-dissociable bonds. Consequently, NS₁ in the nucleolus is not serving as precursor to a ribosomal structural protein.     

Equine arteritis virus-infected cells contain six polyadenylated virus-specific RNAs

Fluorescamine-labeled structural proteins of viruses in the Venezuelan equine encephalitis (VEE) serologic complex were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified [¹⁴C]lysine-labeled capsid protein and El and E2 envelope glycoproteins of TC-83 (serologic subtype-variant I-A) virus were mixed with the corresponding [³H]lysine-labeled structural proteins of TC-83, PTF-39 (I-B), P676 (I-C), 3880 (I-D), Mena II (I-E), Everglades Fe 3–7c (II), Mucambo BeAn 8 (III), and Pixuna BeAr 35645 (IV) viruses and codigested with trypsin. Tryptic peptides were resolved by reverse-phase high-pressure liquid chromatography. The capsid and El proteins of I-A, I-B, I-C, and I-D viruses produced identical or nearly identical tryptic peptide maps, whereas the maps of I-E, II, III, and IV capsid and El proteins were distinct from the maps of the corresponding I-A proteins. The tryptic peptide maps of the type-specific protein, E2, of the various viruses showed the most variation and correlated well with the serologic and genetic homologies determined by oligonucleotide fingerprinting of VEE 42 S RNA. Fluorescamine derivatization of viral proteins was shown to have little or no effect on the specificity of trypsin. Trypsin was shown to cleave these proteins at both lysine and arginine residues.     

Adoptive transfer of immunity against virulent Semliki Forest virus with immune spleen cells from mice infected with avirulent Semliki Forest virus

Summary This paper describes the minimal requirements to protect mice adoptively against challenge with virulent Semliki Forest virus (SFV). Early immune serum, from donor mice infected with an avirulent strain of SFV, contained mainly neutralizing IgM immunoglobulins. More of these antibodies (1.80 PND₅₀ vs 0.06 PND₅₀) were needed to protect recipient mice against intraperitoneal challenge (10 LD₅₀ of SFV) than against subcutaneous challenge (7 LD₅₀). Adoptive transfer experiments indicated that a minimum of 3×10⁷ six-day immune spleen cells were also able to protect recipient mice against intraperitoneal challenge with 10 LD₅₀ of SFV. Treatment of these donor cells with cytotoxic antisera and complement revealed both T- and B-lymphocytes were required for optimum adoptive immunity. Surviving recipients of either immune serum or immune spleen cells developed significantly less neutralizing antibodies than control mice. The lower antibody titres in protected mice might be related to either immune serum or immune spleen cell mediated restriction of virus replication; meaning a reduced antigenic stimulus in these mice compared to control mice.     

Viral RNA synthesis in measles virus-infected cells

Sedimentation analysis of measles virus-specific cytoplasmic RNA at different times after infection revealed several size classes. The three specific components found in greatest amount were estimated to have sedimentation coefficients of 20 S, 27 S, and 35 S. A fourth component, 52 S, sedimented with characteristics of viral RNA. 20 S RNA appeared later than the other size classes, and accounted for an increasingly greater proportion of the total. However, with a temperature-sensitive mutant synthesis of all three major components was turned off simultaneously by transfer of infected cultures from permissive to nonpermissive temperature. Subgenomic-size RNA was found in virion preparations made by undiluted passage, but not after serial passage of diluted inoculum.     

Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis.

The involvement of lipid biosynthesis in the replication of Semliki Forest virus (SFV) in HeLa cells has been analyzed by the use of cerulenin, an inhibitor of lipid synthesis. The presence of this agent from the beginning of infection blocks the appearance of viral proteins. However, when the antibiotic is added at later stages of infection it has no effect on protein synthesis, the cleavage of viral proteins and their acylation by palmitic acid. Cerulenin is a powerful inhibitor of viral RNA synthesis, as analyzed by [3H]uridine incorporation, incorporation of [32P]phosphate into viral replication complexes, or Northern blot analysis of viral RNAs hybridized with minus- or plus-stranded riboprobes. Finally, analysis of phospholipids made in SFV-infected cells indicates that viral infection clearly stimulates the synthesis of phosphatidyl choline and modifies the membrane formed as analyzed by sucrose gradient centrifugation. Cerulenin blocks the synthesis of phospholipids and inhibits the formation of new membranes. These results show that, when the synthesis of lipids is blocked by cerulenin, SFV RNA replication is hampered, suggesting that the synthesis of viral RNAs needs continuous lipid synthesis and membrane formation.     

Asparagine-linked oligosaccharides of Semliki Forest virus grown in mosquito cells

Summary The structure of theN-linked oligosaccharides of Semliki Forest viral glycoproteins produced in infected mosquito cells (C6/36) was investigated by biosynthetic labeling, enzymic deglycosylation using endo--N-acetylglucosaminidases H, D, F/glycopeptidase F, exoglycosidase and analysis of the sugars on Concanavalin A-Sepharose columns and by gel filtration chromatography. The results demonstrated that the glycoproteins decorating the virus shed from infected cells haveN-linked glycans with a trimannosyl core similar to the core glycans produced by vertebrate and yeast cells. However, the E₁ glycoprotein produced by infected C6/36 cells exhibited both a trimannosyl core and a modified trimannosyl core most probably with terminalN-acetylglucosamine. The carbohydrate side chains of Semliki Forest envelope proteins displayed two types of structural heterogeneities existing either at differentN-glycosylation sites as in the case of E₂, or at the sameN-glycosylation site as in the case of E₁. In the presence of 1-deoxymannojirimycin, no structural heterogeneities in the glycan chains were found. This strongly suggests that the glycosylation events that lead to the observed sugar heterogeneities occur in the Golgi membranes.     

Borna disease virus-infected astrocytes function in vitro as antigen-presenting and target cells for virus-specific CD 4-bearing lymphocytes

Summary Astrocytes isolated from the brain of newborn Lewis rats and an astrocytic cell line were susceptible to infection with the neurotropic Borna disease virus in vitro. Since astrocytes also have been found to be infected in vivo it seemed appropriate to test this cell type for interaction with a Borna disease virus-specific CD4⁺ T cell line. Borna disease virus-infected astrocytes were found to be capable of presenting virus-specific antigen to virus-specific T cells in vitro. However, the response was significantly enhanced if the purified 38/39 kDa Borna disease virus-specific protein was added exogenously to the cultures. Beside the function as antigen-presenting cells for various antigens including virus-specific protein and myelin basic protein, persistently infected astrocytes were also found to act as target cells for a CD4⁺ T cell line as shown in conventional⁵¹Cr release assays after induction of MHC class II expression by gamma interferon. Infection of astrocytes alone did not cause expression of this self antigen. It could be shown that the ability of CD4⁺ BDV-specific T cells to mediate lysis was in part dependent on the stage of activation. Lymphocytes activated before testing exerted high lysis after only 4h of coincubation with target cells, whereas resting T cells did not cause significant lysis until 12h of coincubation. The dependence of the interaction between effector and target cells on MHC class II antigen was demonstrated by the finding that antibodies to Ia antigens reduced lysis of target cells.Dedicated to Prof. Dr. Rudolf Rott on the occasion of his 65th birthday.