Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344-->Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala-->Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus

Molecular Genetic Evidence that the Hydrophobic Anchors of Glycoproteins E2 and E1 Interact during Assembly of Alphaviruses

Chimeric alphaviruses in which the 6K and glycoprotein E1 moieties of Sindbis virus are replaced with those of Ross River virus grow very poorly, but upon passage, adapted variants arise that grow >100 times better. We have sequenced the entire domain encoding the E2, 6K, and E1 proteins of a number of these adapted variants and found that most acquired two amino acid changes, which had cumulative effects. In three independent passage series, amino acid 380 of E2, which is in the transmembrane domain, was mutated from the original isoleucine to serine in two instances and to valine once. We have now changed this residue to seven others by site-directed mutagenesis and tested the effects of these mutations on the growth of both the chimera [SIN(RRE1)] and of parental Sindbis. These results indicate that the transmembrane domains of glycoproteins E2 and E1 of alphaviruses interact in a sequence-dependent manner and that this interaction is required for efficient budding and assembly of infectious virions

Mutants of Sindbis Virus I. Isolation and Partial Characterization of 89 New Temperature-Sensitive Mutants

More than 100 new temperature-sensitive mutants of Sindbis virus have been isolated, following mutagenesis with nitrous acid, N-methyl-N’-nitro-N-nitrosoguanidine, Sazacytidine, and 5-fluorouridine. Thirty-six of these mutants synthesize at least 60% as much RNA at the nonpermissive temperature as does the parental strain and are designated RNA+; 23 mutants synthesize between 10 and 60% as much RNA as the parental strain at 40° and are designated RNA±; 30 mutants make less than 10% as much RNA at 40° and are called RNA-. The remaining mutants have not been tested for RNA incorporation. The thermal stability at 56° of most of the mutant particles has been examined. The majority of the RNA+ mutants is more sensitive to heating at 56° than the parental HR strain, and RNA+ mutations appear to reside primarily in genes coding for the structural proteins. Approximately 20% of either RNA± or RNA- mutants are thermosensitive, and these mutations thus appear to reside primarily in genes coding for the nonstructural proteins. Complementation assays have been performed with a number of these mutants and with those of Burge and Pfefferkorn (1966a, b). The existence of three complementation groups among the RNA+ mutants, which appear to encode the three major structural proteins, has been confirmed; no new complementation groups among RNA+ mutants have been identified. A total of four complementation groups has been identified among the RNA- mutants. Thus, Sindbis virus contains at least seven complementation groups

Sequence Analysis of Two Mutants of Sindbis Virus Defective in the Intracellular Transport of Their Glycoproteins

We have sequenced the complementary DNA corresponding to the genes encoding the viral glycoproteins of tslO and ts23, mutants of Sindbis virus defective in the intracellular transport of their glycoproteins, and of revertants of these mutants. These studies have been augmented by direct amino acid sequencing of the amino-terminal regions of the glycoproteins of several virus strains. By comparing the deduced amino acid sequence with that of Sindbis HR virus, the parental strain of these mutants, and with the sequence of the revertants, we found ts23 to have a double mutation in glycoprotein El, while tsl0 was a single mutant in the same glycoprotein. In each case reversion to temperature insensitivity occurred by changes at the same site as the mutation, in two cases restoring the original amino acid and in the third case substituting an homologous amino acid (arginine in place of lysine). The three mutations were far apart from each other in the protein, suggesting that the three-dimensional conformation is very important for the correct migration of the glycoproteins from the rough endoplasmic reticulum to the plasma membrane. The sequence data also reveal that a number of other changes have occurred in the various virus strains during mutagenesis or passage

Mutants of sindbis virus II. Characterization of a maturation-defective mutant, ts103

Mutant ts103 is a minute-plaque former which grows very slowly at any temperature and produces, under optimal conditions, virus yields of 3–10% of those of the parental HR strain. It is slightly temperature sensitive with growth. It reverts at a frequency of 10^(−7) to 10^(−8) to large-plaque, temperature-independent strains and thus is possibly two mutations removed from the large-plaque HR strain of Sindbis virus. The very slow rate of virus production by ts103 appears to be due to a defect in the final stages of virus maturation, the budding of nucleocapsids through the plasma membrane to produce infectious virus. RNA synthesis after infection by the mutant appears to be normal. Nucleocapsids are produced in ts103-infected cells in amounts comparable to that produced in HR-infected cells, although a significant fraction of the mutant nucleocapsids sediment more slowly than HR capsids. Viral hemagglutinin appears in the cell surface earlier in ts103 infection than in HR infection. Electron microscopy of cells infected by ts103 reveals the presence of large amounts of nucleocapsids apparently in the process of budding. Yet the release of mature virus is delayed and the final yield of virus is much reduced in ts103 infection. Furthermore, ts103 budding occurs almost exclusively in virus-specific processes which are quite different in appearance from those found after infection by other strains of Sindbis virus. The virus-like particles produced during infection of chick embryo fibroblasts by this mutant have been examined by sedimentation velocity, isopycnic centrifugation, thermal inactivation at 56°, acrylamide-gel electrophoresis of viral RNA and viral protein, and electron microscopy. Some particles are produced during ts103 infection, which cosediment with Sindbis HR virus at 280 S, and are indistinguishable from HR by a number of other criteria, including isopycnic density, protein composition, sedimentation coefficient of the nucleocapsid, size of RNA, and specific infectivity. However, these 280 S ts103 particles are more sensitive to thermal inactivation at 56° than HR virions. Most of the virus-specific particles produced during ts103 infection sediment faster than 280 S and are heterogeneous in structure. These particles contain more than one nucleocapsid in a single envelope, and the tightness of packing of the nucleocapsids in the envelope is different from particle to particle. This leads to variability in isopycnic density, with particles denser or less dense than HR virus, as well as particles of HR density. In addition, this pleomorphism means that particles containing the same number of nucleocapsids may differ in sedimentation coefficient. These rapidly sedimenting ts103 particles contain small numbers of the nucleocapsids which sediment more slowly than capsids from HR virus, but most of the nucleocapsids are indistinguishable from those of HR. The protein composition of these multicored particles is very similar to that of the HR strain (as examined by acrylamidegel electrophoresis) although they do appear to contain slightly higher ratios of nucleocapsid protein relative to the glycoproteins. These multicored particles are fully infectious with a specific infectivity approaching 100%. The data are all consistent with the following hypothesis: ts103 has an altered nucleocapsid protein. During budding, ts103 capsids interact less strongly with viral glycoproteins in the cell surface than is the case for HR infection. This weakened binding results in a very slow rate of maturation and the production of a large fraction of multiploid particles. In addition some misassembled capsids arise which are unable to mature into normal-sized virions

Expression of the structural proteins of dengue 2 virus and yellow fever virus by recombinant vaccinia viruses

Vaccinia virus recombinants were constructed which contained cDNA sequences encoding the structural region of dengue 2 virus (PR159/S1 strain) or yellow fever virus (17D strain). The flavivirus cDNA sequences were expressed under the control of the vaccinia 7.5k early/late promotor. Cultured cells infected with these recombinants expressed immunologically reactive flavivirus structural proteins, precursor prM and E. These proteins appeared to be cleaved and glycosylated properly since they comigrated with the authentic proteins from dengue 2 virus- and yellow fever virus-infected cells. Mice immunized with the dengue/vaccinia recombinant showed a dengue-specific immune response that included low levels of neutralizing antibodies. Immunization of mice with the yellow fever/vaccinia recombinant was less effective at inducing an immune response to yellow fever virus and in only some of the mice were low titers of neutralizing antibodies produced

Mutants of sindbis virus II. Characterization of a maturation-defective mutant, ts103

Mutant ts103 is a minute-plaque former which grows very slowly at any temperature and produces, under optimal conditions, virus yields of 3–10% of those of the parental HR strain. It is slightly temperature sensitive with growth. It reverts at a frequency of 10^(−7) to 10^(−8) to large-plaque, temperature-independent strains and thus is possibly two mutations removed from the large-plaque HR strain of Sindbis virus. The very slow rate of virus production by ts103 appears to be due to a defect in the final stages of virus maturation, the budding of nucleocapsids through the plasma membrane to produce infectious virus. RNA synthesis after infection by the mutant appears to be normal. Nucleocapsids are produced in ts103-infected cells in amounts comparable to that produced in HR-infected cells, although a significant fraction of the mutant nucleocapsids sediment more slowly than HR capsids. Viral hemagglutinin appears in the cell surface earlier in ts103 infection than in HR infection. Electron microscopy of cells infected by ts103 reveals the presence of large amounts of nucleocapsids apparently in the process of budding. Yet the release of mature virus is delayed and the final yield of virus is much reduced in ts103 infection. Furthermore, ts103 budding occurs almost exclusively in virus-specific processes which are quite different in appearance from those found after infection by other strains of Sindbis virus. The virus-like particles produced during infection of chick embryo fibroblasts by this mutant have been examined by sedimentation velocity, isopycnic centrifugation, thermal inactivation at 56°, acrylamide-gel electrophoresis of viral RNA and viral protein, and electron microscopy. Some particles are produced during ts103 infection, which cosediment with Sindbis HR virus at 280 S, and are indistinguishable from HR by a number of other criteria, including isopycnic density, protein composition, sedimentation coefficient of the nucleocapsid, size of RNA, and specific infectivity. However, these 280 S ts103 particles are more sensitive to thermal inactivation at 56° than HR virions. Most of the virus-specific particles produced during ts103 infection sediment faster than 280 S and are heterogeneous in structure. These particles contain more than one nucleocapsid in a single envelope, and the tightness of packing of the nucleocapsids in the envelope is different from particle to particle. This leads to variability in isopycnic density, with particles denser or less dense than HR virus, as well as particles of HR density. In addition, this pleomorphism means that particles containing the same number of nucleocapsids may differ in sedimentation coefficient. These rapidly sedimenting ts103 particles contain small numbers of the nucleocapsids which sediment more slowly than capsids from HR virus, but most of the nucleocapsids are indistinguishable from those of HR. The protein composition of these multicored particles is very similar to that of the HR strain (as examined by acrylamidegel electrophoresis) although they do appear to contain slightly higher ratios of nucleocapsid protein relative to the glycoproteins. These multicored particles are fully infectious with a specific infectivity approaching 100%. The data are all consistent with the following hypothesis: ts103 has an altered nucleocapsid protein. During budding, ts103 capsids interact less strongly with viral glycoproteins in the cell surface than is the case for HR infection. This weakened binding results in a very slow rate of maturation and the production of a large fraction of multiploid particles. In addition some misassembled capsids arise which are unable to mature into normal-sized virions

Amino-terminal amino acid sequences of structural proteins of three flaviviruses

N-terminal amino acid sequences of structural proteins of three flaviviruses, yellow fever, St. Louis encephalitis, and dengue-2 viruses, have been obtained. The glycoproteins of these three viruses are 52-60% conserved in the region sequenced, depending upon which pair of viruses are compared, and 40% of the amino acids are invariant in all three viruses. Thus, flaviviruses are closely related and have in all probability descended from a common ancestor. Furthermore, residues important in the secondary structure of proteins are conserved, suggesting that the overall conformation of the glycoproteins is the same in all three viruses while considerable variation in the primary sequence can be accommodated. The N-terminal regions of the nucleocapsid proteins of yellow fever and St. Louis encephalitis viruses show markedly less homology (25%) and this region is highly basic with one-quarter (yellow fever) or one-third (St. Louis encephalitis) of the residues being lysine or arginine. N-terminal sequences for the M protein of yellow fever and for NV2(GP19) of St. Louis encephalitis viruses are also reported

Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution

The sequence of the entire RNA genome of the type flavivirus, yellow fever virus, has been obtained. Inspection of this sequence reveals a single long open reading frame of 10,233 nucleotides, which could encode a polypeptide of 3411 amino acids. The structural proteins are found within the amino-terminal 780 residues of this polyprotein; the remainder of the open reading frame consists of nonstructural viral polypeptides. This genome organization implies that mature viral proteins are produced by posttranslational cleavage of a polyprotein precursor and has implications for flavivirus RNA replication and for the evolutionary relation of this virus family to other RNA viruses