Frameshifting in Alphaviruses: A Diversity of 3′ Stimulatory Structures

Programmed ribosomal frameshifting allows the synthesis of alternative, N-terminally coincident, C-terminally distinct proteins from the same RNA. Many viruses utilize frameshifting to optimize the coding potential of compact genomes, to circumvent the host cell's canonical rule of one functional protein per mRNA, or to express alternative proteins in a fixed ratio. Programmed frameshifting is also used in the decoding of a small number of cellular genes. Recently, specific ribosomal − 1 frameshifting was discovered at a conserved U_UUU_UUA motif within the sequence encoding the alphavirus 6K protein. In this case, frameshifting results in the synthesis of an additional protein, termed TF (TransFrame). This new case of frameshifting is unusual in that the − 1 frame ORF is very short and completely embedded within the sequence encoding the overlapping polyprotein.The present work shows that there is remarkable diversity in the 3′ sequences that are functionally important for efficient frameshifting at the U_UUU_UUA motif. While many alphavirus species utilize a 3′ RNA structure such as a hairpin or pseudoknot, some species (such as Semliki Forest virus) apparently lack any intra-mRNA stimulatory structure, yet just 20 nt 3′-adjacent to the shift site stimulates up to 10% frameshifting. The analysis, both experimental and bioinformatic, significantly expands the known repertoire of − 1 frameshifting stimulators in mammalian and insect systems.     

Functional Characterization of the Alphavirus TF Protein

Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: capsid, pE2, 6K, and E1. However, bioinformatic analyses of alphavirus genomes (A. E. Firth, B. Y. Chung, M. N. Fleeton, and J. F. Atkins, Virol. J. 5:108, 2008) suggested that a ribosomal frameshifting event occurs during translation of the alphavirus structural polyprotein. Specifically, a frameshift event is suggested to occur during translation of the 6K gene, yielding production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the −1 open reading frame (ORF). Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.     

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.     

Biological activity of invitro synthesised protein: Binding of Semliki Forest virus capsid protein to the large ribosomal subunit

Cell-free translation of the Semliki Forest virus-specific 26S RNA yielded primarily capsid protein. After treatment of the protein synthesising reaction with 25 mM EDTA, the capsid protein cosedimented with the large ribosomal subunit in sucrose gradients, and banded with the subunit at a density of 1.54 gm/cm³ in CsCl. Exposure to 0.5 M KCl released the protein from the subunit. Similar binding of the virus capsid protein to the large ribosomal subunit has been observed in infected HeLa cells, although its function is not clear. The nonstructural proteins, which are the major products translated from the virion 42S RNA, did not associate with sedimenting structures.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Alphavirus 6K proteins form ion channels

Ross River virus and Barmah Forest virus are Australian arboviruses of the Alphavirus genus. Features of alphavirus infection include an increased permeability of cells to monovalent cations followed by virion budding. Virally encoded ion channels are thought to have a role in these processes. In this paper, the 6K proteins of Ross River virus and Barmah Forest virus are shown to form cation-selective ion channels in planar lipid bilayers. Using a novel purification method, bacterially expressed 6K proteins were inserted into bilayers with a defined orientation (i.e. N-terminal cis, C-terminal trans). Channel activity was reversibly inhibited by antibodies to the N and C termini of 6K protein added to the cis and trans baths, respectively. Channel conductances varied from 40-800 picosiemens, suggesting that the protein is able to form channels with a range of possible oligomerization states.     

Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Background

Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses.

Methodology/Principal Findings

Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized.

Conclusions/Significance

Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.     

The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy

Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed -1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.     

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.     

Tryptic peptide analysis on nonstructural and structural precursor proteins from Semliki Forest virus mutant-infected cells.

Analysis of [35S]methionine-labeled tryptic peptides of the large proteins induced by temperature-sensitive mutants of Semliki Forest virus was carried out. The 130,000-molecular-weight protein induced by ts-2 and ts-3 mutants contained the peptides of capsid protein and of both major envelope proteins E1 and E2. The ts-3-induced protein with molecular weight of 97,000 contained peptides of the capsid and envelope protein E2 but not those of E1. Two proteins with molecular weights of 78,000 and 86,000 from ts-1-infected cells did not contain the peptides of the virion structural proteins. They are evidently expressions of the nonstructural part of the 42S RNA genome of Semliki Forest virus.     

Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes

Background

Although many vaccinia virus proteins have been identified and studied in detail, only a few studies have attempted a comprehensive survey of the protein composition of the vaccinia virion. These projects have identified the major proteins of the vaccinia virion, but little has been accomplished to identify the unknown or less abundant proteins. Obtaining a detailed knowledge of the viral proteome of vaccinia virus will be important for advancing our understanding of orthopoxvirus biology, and should facilitate the development of effective antiviral drugs and formulation of vaccines.

Results

In order to accomplish this task, purified vaccinia virions were fractionated into a soluble protein enriched fraction (membrane proteins and lateral bodies) and an insoluble protein enriched fraction (virion cores). Each of these fractions was subjected to further fractionation by either sodium dodecyl sulfate-polyacrylamide gel electophoresis, or by reverse phase high performance liquid chromatography. The soluble and insoluble fractions were also analyzed directly with no further separation. The samples were prepared for mass spectrometry analysis by digestion with trypsin. Tryptic digests were analyzed by using either a matrix assisted laser desorption ionization time of flight tandem mass spectrometer, a quadrupole ion trap mass spectrometer, or a quadrupole-time of flight mass spectrometer (the latter two instruments were equipped with electrospray ionization sources). Proteins were identified by searching uninterpreted tandem mass spectra against a vaccinia virus protein database created by our lab and a non-redundant protein database.

Conclusion

Sixty three vaccinia proteins were identified in the virion particle. The total number of peptides found for each protein ranged from 1 to 62, and the sequence coverage of the proteins ranged from 8.2% to 94.9%. Interestingly, two vaccinia open reading frames were confirmed as being expressed as novel proteins: E6R and L3L.     

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.     

Nairovirus RNA sequences expressed by a Semliki Forest virus replicon induce RNA interference in tick cells

We report the successful infection of the cell line ISE6 derived from Ixodes scapularis tick embryos by the tick-borne Hazara virus (HAZV), a nairovirus in the family Bunyaviridae. Using a recombinant Semliki Forest alphavirus replicon that replicates in these cells, we were able to inhibit replication of HAZV, and we showed that this blockage is mediated by the replication of the Semliki Forest alphavirus replicon; the vector containing the HAZV nucleoprotein gene in sense or antisense orientation efficiently inhibited HAZV replication. Moreover, expression of a distantly related nucleoprotein gene from Crimean-Congo hemorrhagic fever nairovirus failed to induce HAZV silencing, indicating that the inhibition is sequence specific. The resistance of these cells to replicate HAZV correlated with the detection of specific RNase activity and 21- to 24-nucleotide-long small interfering RNAs. Altogether, these results strongly suggest that pathogen-derived resistance can be established in the tick cells via a mechanism of RNA interference.     

A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.     

A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal

A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.     

Prefusion rearrangements resulting in fusion Peptide exposure in Semliki forest virus

Semliki Forest virus (SFV), like many enveloped viruses, takes advantage of the low pH in the endosome to convert into a fusion-competent configuration and complete infection by fusion with the endosomal membrane. Unlike influenza virus, carrying an N-terminal fusion peptide, SFV represents a less-well understood fusion principle involving an endosequence fusion peptide. To explore the series of events leading to a fusogenic configuration of the SFV, we exposed the virus to successive acidification, mimicking endosomal conditions, and followed structural rearrangements at probed sensor surfaces. Thus revealed, the initial phase involves a transient appearance of a non-linear neutralizing antibody epitope in the fusion protein, E1. Concurrent with the disappearance of this epitope, a set of masked sequences in proteins E1 and E2 became exposed. When pH reached 6.0-5.9 the virion transformed into a configuration of enlarged diameter with the fusion peptide optimally exposed. Simultaneously, a partly hidden sequence close to the receptor binding site in E2 became fully uncovered. At this presumably fusogenic stage, maximally 80 fusion peptide-identifying antibody Fab fragments could be bound per virion, i.e. one ligand per three copies of the fusion protein. The phenomena observed are discussed in terms of alphavirus structure and reported functional domains.     

Proteomic analysis of the EhV-86 virion

Background

Emiliania huxleyi virus 86 (EhV-86) is the type species of the genus Coccolithovirus within the family Phycodnaviridae. The fully sequenced 407,339 bp genome is predicted to encode 473 protein coding sequences (CDSs) and is the largest Phycodnaviridae sequenced to date. The majority of EhV-86 CDSs exhibit no similarity to proteins in the public databases.

Results

Proteomic analysis by 1-DE and then LC-MS/MS determined that the virion of EhV-86 is composed of at least 28 proteins, 23 of which are predicted to be membrane proteins. Besides the major capsid protein, putative function can be assigned to 4 other components of the virion: two lectin proteins, a thioredoxin and a serine/threonine protein kinase.

Conclusion

This study represents the first steps toward the identification of the protein components that make up the EhV-86 virion. Aside from the major capsid protein, whose function in the virion is well known and defined, the nature of the other proteins suggest roles involved with viral budding, caspase activation, signalling, anti-oxidation, virus adsorption and host range determination.     

Structural proteins of Western equine encephalitis virus: amino acid compositions and N-terminal sequences

The structural proteins of Western equine encephalitis virus, a member of the alphavirus group, have been characterized by the determination of their amino acid compositions and by N-terminal sequence analysis. More than 60 residues of the N-terminal sequences of each of the envelope glycoproteins have been determined. A comparison of these sequences with the previously determined sequences of two related alphaviruses. Sindbis virus and Semliki Forest virus, strongly supports the view that all three viruses have evolved from a common ancestor and provides information on the pattern of this evolution. The analysis of the capsid proteins of Western equine encephalitis virus shows that the nucleocapsid of this virus can accommodate a considerable degree of variability in its protein component and that at least some regions of alphavirus capsid proteins show more extensive differences between different viruses than do the envelope glycoproteins.     

Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit.

The alphavirus Semliki Forest virus (SFV) matures by budding at the cell surface. This process is driven by interactions of its membrane protein heterodimer E2-E1 and the nucleocapsid. The virus penetrates into new cells by an E1-mediated membrane fusion event. The E1 subunit has a short, strongly positively charged cytoplasmic tail peptide (Arg-Arg) which is very conserved among different alphavirus E1 proteins. In this work, we have used in vitro mutagenesis of a full-size cDNA clone of SFV to study the role of the tail peptide of the E1 subunit in virus budding and fusion processes in baby hamster kidney cell culture. Our results suggest that the E1 tail plays no major role in SFV multiplication in animal cell culture.