Specific binding of host cell proteins to the 3''-terminal stem-loop structure of rubella virus negative-strand RNA.

At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.     

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.     

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.     

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.     

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.     

Mosquito homolog of the La autoantigen binds to Sindbis virus RNA.

We have isolated a 50-kDa mosquito protein that binds with high affinity to a riboprobe representing the 3' end of the minus strand of Sindbis virus RNA. The isolated protein has been used to obtain cDNA clones encoding this protein that have been sequenced and used to express the protein in large amounts. Sequence comparisons make clear that this protein is the mosquito homolog of the La autoantigen. The N-terminal half of the protein shares considerable sequence identity with the human La protein, the rat La protein, and the recently identified Drosophila melanogaster homolog. There is one stretch of 100 amino acids in the N-terminal domain in which 48 residues are identical in all four proteins. In contrast, the C-terminal domain of the mosquito protein shares little identity with any of the other three proteins. We have also shown that the mosquito protein, the human protein, and a putative chicken homolog of the La protein cross-react immunologically and, thus, all share antigenic epitopes. The mosquito La protein is primarily nuclear in location, but significant amounts are present in the cytoplasm, as is the case for the La proteins of other species. The equilibrium constant for the binding of the expressed mosquito La protein to the Sindbis virus riboprobe is 15.4 nM, and thus the affinity of binding is high enough to be physiologically relevant. Furthermore, the conservation of this protein in the animal kingdom may be significant, because Sindbis virus utilizes mosquitoes, birds, and mammals as hosts. We propose that the interactions we observe between the La protein and toes, birds, and mammals as hosts. We propose that the interactions we observe between the La protein and a putative promoter in the Sindbis virus genome are significant for Sindbis virus RNA replication.     

Aura virus structure suggests that the T=4 organization is a fundamental property of viral structural proteins

Aura and Sindbis viruses are closely related alphaviruses. Unlike other alphaviruses, Aura virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virus particles. Previous studies on negatively stained Aura virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 A, respectively, and compared with reconstructions of Sindbis virus and Ross River virus particles. Aura virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaviruses. The morphology of Aura virus glycoprotein spikes closely resembles that of Sindbis virus spikes and is detectably different from that of Ross River virus spikes. Thus, some aspects of the surface structure of members of the Sindbis virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River virus lineage.     

BHK cell proteins that bind to the 3' stem-loop structure of the West Nile virus genome RNA.

The first 83 3' nucleotides of the genome RNA of the flavivirus West Nile encephalitis virus (WNV) form a stable stem-loop (SL) structure which is followed in the genome by a smaller SL. These 3' structures are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and as such might specifically bind to cellular or viral proteins. Cellular proteins from uninfected and WNV-infected BHK-21 S100 cytoplasmic extracts formed three distinct complexes with the WNV plus-strand 3' SL [(+)3'SL] RNA in a gel mobility shift assay. Subsequent competitor gel shift analyses showed that two of these RNA-protein complexes, complexes 1 and 2, contained cell proteins that specifically bound to the WNV (+)3'SL RNA. UV-induced cross-linking and Northwestern blotting analyses detected WNV (+)3'SL RNA-binding proteins of 56, 84, and 105 kDa. When the S100 cytoplasmic extracts were partially purified by ion-exchange chromatography, a complex that comigrated with complex 1 was detected in fraction 19, while a complex that comigrated with complex 2 was detected in fraction 17. UV-induced cross-linking experiments indicated that an 84-kDa cell protein in fraction 17 and a 105-kDa protein in fraction 19 bound specifically to the WNV (+)3'SL RNA. In addition to binding to the (+)3'SL RNA, the 105-kDa protein bound to the SL structure located at the 3' end of the WNV minus-strand RNA. Initial mapping studies indicated that the 84- and 105-kDa proteins bind to different regions of the (+)3'SL RNA. The 3'-terminal SL RNA of another flavivirus, dengue virus type 3, specifically competed with the WNV (+)3'SL RNA in gel shift assays, suggesting that the host proteins identified in this study are flavivirus specific.     

Binding of Sindbis Virus to Cell Surface Heparan Sulfate

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.     

Dependence of the encapsidation function of the adenovirus L1 52/55-kilodalton protein on its ability to bind the packaging sequence

The adenovirus IVa2 and L1 52/55-kDa proteins are involved in the assembly of new virus particles. Both proteins bind to the packaging sequence of the viral chromosome, and the lack of expression of either protein results in no virus progeny: the absence of the L1 52/55-kDa protein leads to formation of only empty capsids, and the absence of the IVa2 protein results in no capsid assembly. Furthermore, the IVa2 and L1 52/55-kDa proteins interact with each other during adenovirus infection. However, what is not yet clear is when and how this interaction occurs during the course of the viral infection. We defined the domains of the L1 52/55-kDa protein required for interaction with the IVa2 protein, DNA binding, and virus replication by constructing L1 52/55-kDa protein truncations. We found that the N-terminal 173 amino acids of the L1 52/55-kDa protein are essential for interaction with the IVa2 protein. However, for both DNA binding and complementation of the pm8001 mutant virus, which does not express the L1 52/55-kDa protein, the amino-terminal 331 amino acids of the L1 52/55-kDa protein are necessary. These results suggest that the production of infectious virus particles depends on the ability of the L1 52/55-kDa protein to bind to DNA.     

A regulatory cis element and a specific binding factor involved in the mitogenic control of murine ribosomal protein L32 translation.

The mRNA encoding ribosomal protein L32 redistributes from untranslated subribosomal particles into polysomes after mitogenic activation of quiescent T-lymphocytes and fibroblasts. To identify the regions of the L32 mRNA which are important in regulating its cytoplasmic location we constructed a plasmid containing the murine L32 cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat promoter and introduced this construct into murine 3T3 fibroblasts. The mRNA transcribed from the RSV-L32 construct redistributed from subribosomal particles into polysomes in response to mitogenic activation in a manner similar to endogenous L32 mRNA. A conserved polypyrimidine region present at the 5' terminus of all ribosomal protein mRNAs is required for translational regulation of L32 mRNA since deletion of this sequence resulted in a mRNA that was not sequestered in subribosomal particles in quiescent cells. A radioactive RNA probe containing the first 34 nucleotides of the L32 5'-untranslated region, including the polypyrimidine region, specifically interacted with a protein of about 56 kDa. This protein did not bind detectably to RNA probes lacking the polypyrimidine sequence. Binding activity was similar in protein extracts made from resting and activated cells, suggesting that binding of the 56-kDa protein as measured in this assay is not regulated. This protein is a member of what may be an emerging family of polyribopyrimidine-binding proteins with diverse biochemical functions.     

Microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell.

The microviscosities of the hydrophobic regions of the membranes of intact Semliki forest and Sindbis viruses grown on BHK-21 cells, of liposomes derived from the extracted viral lipids, and of protease-treated virions were measured by fluorescence depolorization using the fluorescence probe 1, 6-diphenyl-1,3,5-hexatriene. The intact virus membranes were found to have a higher microviscosity than did virus-derived liposomes, indicating the viral envelope proteins contribute to microviscosity. However, protease-treated virus, devoid of protruding spikes but with residual lipophilic peptide tails, was found to have a microviscosity more similar to that of the intact virus than to that of protein-free liposomes. Sindbis virus grown in BHK-21 cells at 37 C had a much higher microviscosity than did Sindbis virus grown on Aedes albopicuts cells at 22 C. Sindbis virus grwon in A. albopictus and BHK-21 cells also gave higher microviscosity values than did the intact host cells. These data indicate that both the virion proteins and the cellular lipids selected during viral growth and maturation contribute to the increased microviscosity of togavirus membranes.     

Defined mutations in the 5'' nontranslated sequence of Sindbis virus RNA.

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant.     

Differential cholesterol binding by class II fusion proteins determines membrane fusion properties

The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.     

An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication.

Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.     

Common and distinct regions of defective-interfering RNAs of Sindbis virus

Defective-interfering (DI) particles are helper-dependent deletion mutants which interfere specifically with the replication of the homologous standard virus. Serial passaging of alphaviruses in cultured cells leads to the accumulation of DI particles whose genomic RNAs are heterogeneous in size and sequence composition. In an effort to examine the sequence organization of an individual DI RNA species generated from Sindbis virus, we isolated and sequenced a representative cDNA clone derived from a Sindbis DI RNA population. Our data showed that: (i) the 3' end of the DI RNA template was identical to the 50 nucleotides at the 3' end of the standard RNA; (ii) the majority (75%) of the DI RNA template was derived from the 1,200 5'-terminal nucleotides of the standard RNA and included repeats of these sequences; and (iii) the 5' end of the DI RNA template was not derived from the standard RNA, but is nearly identical to a cellular tRNAAsp (S. S. Monroe and S. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 80:3279-3283, 1983). We have also utilized restriction fragments from cloned DNAs to probe by blot hybridization for the presence of conserved sequences in several independently derived DI RNA populations. These studies indicated that: (i) a 51-nucleotide conserved sequence located close to the 5' end of several alphavirus RNAs was most likely retained in the DI RNAs; (ii) the junction region containing the 5' end of the subgenomic 26S mRNA was deleted from the DI RNAs; and (iii) the presence of tRNAAsp sequences was a common occurrence in Sindbis virus DI RNAs derived by passaging in chicken embryo fibroblasts.