The proteolytic cleavage of PE2 to envelope glycoprotein E2 is not strictly required for the maturation of Sindbis virus.

The ionophore monensin has been shown previously to block the maturation of Sindbis virus as well as prevent the cleavage of pE2 to E2 when applied to cells in high concentration. We found that a moderate dose of monensin reduced virus titer and inhibited the cleavage of pE2 to E2. Under these conditions, pE2 appeared on the cell surface in a form susceptible to lactoperoxidase-mediated iodination. This pE2 was incorporated into virions, replacing E2. PE2-containing virions had a normal PFU-to-particle ratio, cosedimented with normal virus, and retained a normal morphology when negatively stained preparations were examined by electron microscopy. We conclude that the cleavage of pE2 to form E2 is not an absolute prerequisite for virus maturation. Recently, Russell et al. have reached a similar conclusion (D. L. Russell, J. M. Dalrymple, and R. E. Johnston, J. Virol. 63:1619-1629, 1989).     

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype.

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.     

The Furin Protease Cleavage Recognition Sequence of Sindbis Virus PE2 Can Mediate Virion Attachment to Cell Surface Heparan Sulfate

Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences.     

The host range phenotype displayed by a Sindbis virus glycoprotein variant results from virion aggregation and retention on the surface of mosquito cells

The Sindbis virus variant NE2G216 is a PE2-containing host range mutant that is growth restricted in cultured mosquito cells (C6/36) due to inefficient release of virions from this cell type. The maturation defect of NE2G216 has been linked to the structures of N-linked oligosaccharides synthesized by arthropod cells. Analysis of C6/36 cells infected with NE2G216 by transmission electron microscopy revealed the presence of dense virus aggregates within cytoplasmic vacuoles and virus aggregates adhered to the cell surface. The virus aggregation phenotype of NE2G216 was reproduced in vertebrate cells (Pro-5) by the addition of 1-deoxymannojirimycin, an inhibitor of carbohydrate processing which limits the processing of N-linked oligosaccharides to structures that are structurally similar, albeit not identical, to those synthesized in C6/36 cells. We conclude that defective maturation of NE2G216 in mosquito cells is due to virion aggregation and retention on the cell surface and that this phenotype is directly linked to the carbohydrate-processing properties of these cells.     

Defective transport of Sindbis virus glycoproteins in End4 mutant Chinese hamster ovary cells.

Mutant V.24.1, a temperature-sensitive derivative of Chinese hamster ovary cells, defines the End4 complementation group of mutants selected for resistance to protein toxins and has defective lysosomes at the restrictive temperature (P. A. Colbaugh, M. Stookey, and R. K. Draper, J. Cell Biol. 108:2211-2219, 1989). We have investigated the biosynthesis of Sindbis virus envelope glycoproteins in V.24.1 cells. When the cells were infected at the restrictive temperature, the envelope glycoproteins E1 and E2 were undetectable on the cell surface and proteolytic processing of the precursor protein pE2 to envelope protein E2 did not occur. Protein retained intracellularly was sensitive to endoglycosidase H and, by immunofluorescence localization, appeared to accumulate in the endoplasmic reticulum. We conclude that the genetic defect in V.24.1 cells impairs the transport of Sindbis virus glycoproteins, apparently at the level of export from the endoplasmic reticulum.     

Antiviral activity of alpha interferon in Sindbis virus-infected cells is restored by anti-E2 monoclonal antibody treatment.

Pretreatment of AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2 (AT3-bcl-2 cells) with alpha interferon (IFN-alpha) affected replication of a virulent strain of Sindbis virus (SV) but did not protect against virus-induced cell death. Treatment of cells with IFN-alpha late during infection affected ongoing SV replication very little. Previous studies have shown that cross-linking of the viral glycoprotein E2 with antibody delays the inhibition of K+ influx by improving the function of Na+K+ATPase and the Na(+)-K(+)-2Cl-cotransport system in SV-infected cells (P. Després, J. W. Griffin, and D. E. Griffin, J. Virol. 69:7006-7014, 1995). In these studies, we have shown that treatment of infected cells with anti-E2 monoclonal antibody also restored the ability of IFN-alpha to induce antiviral activity in infected cells late during infection. The very low rate of virus release in SV-infected cells treated simultaneously with anti-E2 monoclonal antibody and IFN-alpha was postulated to be linked to inhibition of virus maturation. Synergistic effects of antibody and IFN-alpha are likely to be important for control of SV replication in vivo.     

Role of glycoprotein PE2 in formation and maturation of the Sindbis virus spike.

Sindbis virus envelope assembly is a multistep process resulting in the maturation of a rigid, highly ordered T=4 icosahedral protein lattice containing 80 spikes composed of trimers of E1-E2 heterodimers. Intramolecular disulfide bonds within E1 stabilize E1-E1 associations required for envelope formation and maintenance of the envelope's structural integrity. The structural integrity of the envelope protein lattice is resistant to reduction by dithiothreitol (DTT), indicating that E1 disulfides which stabilize structural domains become inaccessible to DTT at some point during virus maturation. The development of E1 resistance to DTT occurs prior to the completion of E1 folding and is temporally correlated with spike assembly in the endoplasmic reticulum. From these data we have predicted that in the final stages of spike assembly, E1 intramolecular disulfides, which stabilize the structural integrity of the envelope protein lattice, are buried within the spike and become inaccessible to the reductive activity of DTT. The spike is formed prior to the completion of E1 folding, and we have suggested that PE2 (the precursor to E2) may play a critical role in E1 folding after PE2-E1 oligomer formation has occurred. In this study we have investigated the role of PE2 in E1 folding, oligomer formation, and development of E1 resistance to both protease digestion and reduction by DTT by using a Sindbis virus replicon (SINrep/E1) which allows for the expression of E1 in the presence of truncated PE2. Through pulse-chase analysis of both Sindbis virus- and SINrep/E1-infected cells, we have determined that the folding of E1 into a trypsin-resistant conformation and into its most compact and stable form is not dependent upon association of E1 with PE2. However, E1 association with PE2 is required for oligomer formation, the export of E1 from the endoplasmic reticulum, and E1 acquisition of resistance to DTT.     

Requirement of cell nucleus for Sindbis virus replication in cultured Aedes albopictus cells.

The ability of Sindbis virus to grow in enucleated BHK-21 (vertebrate) and Aedes albopictus (invertebrate) cells was tested to determine the dependence of this virus upon nuclear function in these two phylogenetically unrelated hosts. Although both cell types could be demonstrated to produce viable cytoplasts (enucleated cells) which produced virus-specific antigen subsequent to infection. BHK cytoplasts produced a significant number of progeny virions, whereas mosquito cytoplasts did not. The production of vesicular stomatitis virus in mosquito cells was not significantly reduced by enucleation. That such a host function was not essential for vesicular stomatitis virus growth in insect cells is supported by the observation that the production of this virus by mosquito cells is not actinomycin D sensitive. This result agrees with a previously published report in which it was shown that Sindbis virus maturation in invertebrate cells is inhibited by actinomycin D, indicating a possible requirement for host cell nuclear function (Scheefers-Borchel et al., Virology, 110:292-301, 1981).     

A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A and alphaviruses fails to cleave Sindbis virus glycoprotein PE2.

RPE.40, a mutant CHO-K1 strain selected for resistance to Pseudomonas exotoxin A, is defective in the production of infectious alphaviruses, although viruses are taken in and processed normally (J. M. Moehring and T. J. Moehring, Infect. Immun. 41:998-1009, 1983). To determine the cause of this defect, the synthesis of Sindbis virus proteins was examined. RPE.40 cells produced and glycosylated structural glycoprotein precursors PE2 and immature E1 normally. Mature E1 was formed, but PE2 was not cleaved to E2 and E3. PE2 instead was modified to a higher-molecular-weight form (PE2') in which the high-mannose oligosaccharides were processed to the complex form without proteolytic cleavage. The data suggest that the cleavage which produces E2 occurs within the trans-Golgi or in post-Golgi elements and is closely associated with the addition of sialic acid residues to the asparagine-linked oligosaccharides. RPE.40 cells make and release noninfectious Sindbis virions that contain PE2' and no detectable E2. These virions can be converted to an infectious form by treatment with trypsin. A defect in an intracellular endopeptidase activity in RPE.40 cells is postulated. Comparison of two Sindbis virus strains showed that the requirement for E2 in the virion to ensure infectivity is strain specific.     

Effect of low-NaCl medium on the envelope glycoproteins of Sindbis virus

Lowering the NaCl concentration of the medium inhibits the release of Sindbis virus from infected chicks cells at a stage after the nucleocapsids have bound to the membranes of the infected cells. The failure of trypsin treatment to release the inhibited virus and the ratio of the proteins in the inhibited cells make it seem likely that the inhibited virus is all intracellular. Experiments using antisera specific for E1 and E2, the envelope glycoproteins of Sindbis, suggest that the inhibitory effect of low-salt medium is mediated through an effect on E2. Lactoperoxidase radioiodination experiments indicate that, even when cleaved from PE2, E2 is not exposed on the surface of low-NaCl-treated chick cells.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Effects of l-Glutamine Deprivation on Growth of HVJ (Sendai Virus) in BHK Cells

l-Glutamine requirement for viral maturation was found in BHK-HVJ cells, a cell line of baby hamster kidney cells persistently infected with HVJ (Sendai virus). Synthesis of envelope protein in BHK-HVJ cells was markedly suppressed by deprivation of l-glutamine, whereas development of nucleocapsid (S) antigen was less affected. More detailed examination of this phenomenon was carried out by using a cytolytic system. Growth of HVJ in BHK cells cultured in media deprived of various amino acids was investigated, and omission of l-glutamine from culture medium resulted in a marked inhibitory effect on the release of infectious virus and synthesis of envelope protein, although synthesis of virus-specific RNA and nucleocapsid antigen in the cells was readily detected. When l-glutamine was restored to the culture medium, infectious virus and envelope protein could be detected. l-Glutamic acid, l-aspartic acid, or l-alanine could be substituted for l-glutamine. Effects of l-glutamine deprivation on HVJ growth in several other cells were also investigated. The growth of HVJ in the cells other than BHK and FL cells was not suppressed by lack of l-glutamine. Growth of Sindbis virus in BHK cells was also markedly retarded in the absence of l-glutamine.     

Envelope antigens of Sindbis virus in cells infected with temperature-sensitive mutants.

Indirect fluorescent-antibody studies of living and fixed chick cells infected with temperature-sensitive mutants of Sindbis virus suggest that functional envelope glycoprotein E1 must be inserted through the plasma membrane before E2. PE2 and E2 do not affect the insertion of E1. The experiments also suggest that normal PE2, a glycosylated precursor to E2, reacts with anti-E2 serum; the abnormal PE2 made by a temperature-sensitive PE2 cleavage-defective mutant did not. Abnormal E1 proteins made by E1-defective mutants also failed to react with anti-E1 serum.     

Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55(Gag) elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.     

Divergent envelope E2 alphavirus sequences spanning amino acids 297 to 352 induce in mice virus-specific protective immunity and antibodies with complement-mediated cytolytic activity.

We have proposed a general algorithm for identification of potential immunoprotective domains (cassettes) on the envelope E2 polypeptide of alphaviruses (H. Grosfeld, B. Velan, M. Leitner, S. Cohen, S. Lustig, B.E. Lachmi, and A. Shafferman, J. Virol. 63:3416-3422, 1989). To assess the generality of our approach, we compared analogous E2 cassettes from Sindbis virus (SIN) and Semliki Forest virus (SFV), two alphaviruses which are philogenetically very remote. The antigenically distinct SFV E2 and SIN E2 cassettes exhibit comparable immunological characteristics. Most significantly, the SIN E2 LMN cassette cluster (E2 amino acids 297 to 352 fused to beta-galactosidase), like the analogous SFV E2 LMN cassettes, elicited high titers of antivirus antibodies in mice and proved to be highly effective in protection against lethal challenge. Mice immunized with SIN E2 LMN were completely protected against intracerebral challenge of 10 to 100 50% lethal doses of different neurovirulent SIN strains. Anti-SIN LMN antibodies, like anti-SFV LMN antibodies, lacked in vitro neutralizing activity, yet both exerted protection against homologous challenge upon transfer to mice. The two antibody preparations exhibited virus-specific complement-mediated cytolysis of cells infected with the homologous but not heterologous virus. These results suggest a possible mechanism for virus-specific E2 LMN-induced protection and demonstrate the generality of our methodology for deciphering immunogenic and protective domains in alphavirus systems. Results suggest also that the E2 LMN sequence of any given alphavirus should be considered as a component of a synthetic vaccine against that specific virus.     

The amino-terminal residue of Sindbis virus glycoprotein E2 influences virus maturation, specific infectivity for BHK cells, and virulence in mice.

The E2 glycoprotein of Sindbis virus is synthesized as a precursor, PE2, which is cleaved by furin or a furin-like host cell protease at a late stage of maturation. The four-residue PE2 cleavage signal conforms to the basic amino acid-X-basic-basic motif which is present in many other viral and cellular glycoproteins which are processed by the cellular enzyme(s). In this report, we present evidence that the amino acid which immediately follows the signal, the N-terminal residue of E2, can influence protease recognition, binding, and/or cleavage of PE2. Constructs encoding nine different amino acids at E2 position 1 (E2 1) were produced by site-directed mutagenesis of the full-length cDNA clone of our laboratory strain of Sindbis virus AR339 (pTRSB). Viruses derived from clones encoding Arg (TRSB), Asp, Ser, Phe, His, and Asn in a nonglycosylated form at E2 1 contained predominantly E2. Viruses encoding Ile, Leu, or Val at E2 1 contained the uncleaved form of PE2. The specific infectivity of TRSB (E2 Arg-1) for baby hamster kidney (BHK-21) cells was from 5- to greater than 100-fold higher than those of isogenic constructs with other residues at E2 1, suggesting that E2 Arg-1 represents a BHK-21 cell adaptive mutation in our laboratory strain. In newborn CD-1 mice, TRSB was more virulent than the PE2-containing viruses but less virulent than other PE2-cleaving viruses with alternative amino acids at E2 1. These results indicate that in TRSB, E2 Arg-1 increased the efficiency of virus-cell interactions in cultured BHK-21 cells but simultaneously decreased the ability of virus to mediate in vivo virus-cell interactions critical for the induction of disease. This suggests that the N terminus of E2 may participate in or be associated with virion domains which mediate these viral functions.     

Interaction of Sindbis virus glycoproteins during morphogenesis.

In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell.     

Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto 1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low-pH-dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto 1101 virus. These results demonstrate that acylation of the transmembrane domain of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity.