Infection of neuroblastoma cells by Semliki Forest virus. The interference of viral capsid protein with the binding of host messenger RNAs into initiation complexes is the cause of the shut-off of host protein synthesis

From ribosomal washes of neuroblastoma cells infected with Semliki Forest virus (SFV) a protein of Mr 33000 was purified, which comigrated with the viral capsid protein on sodium dodecyl sulfate/polyacrylamide gels and was recognized by antibodies against the capsid protein of SFV. This protein selectively inhibits the translation of host and early viral 42S mRNA in vitro, but has no effect on late viral 26S and encephalomyocarditis virus mRNA translation. Eukaryotic initiation factor 4B and cap-binding protein restore the translation of host and 42S mRNA to control levels. The capsid protein specifically prevents the binding of host mRNA into 80S initiation complexes, but has no effect on that of late viral mRNA. We propose that the capsid protein is the component responsible for the shut-off of host protein synthesis in SFV-infected cells and for the decreased translational activity of the crude ribosomal washes from these cells.     

Semliki forest virus capsid protein inhibits the initiation of translation by upregulating the double-stranded RNA-activated protein kinase (PKR)

We investigated the possible translational role which elevated concentrations of highly purified Semliki Forest virus (SFV) capsid (C)-protein molecules may play in a cell-free translation system. Here we decomonstrate that in the absence of double-stranded RNA high concentrations of C protein triggered the phosphorylation of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. Activated PKR in turn phosphorylated its natural substrate, the subunit of eukaryotic initiation factor 2 (eIF-2), thereby inhibiting initiation of host cell translation. These findings were further strengthened by experiments showing that during natural infection with SFV the maximum phosphorylation of PKR coincided with the maximum synthesis of C protein 4–9 hours post infection. Thus, our results demonstrate that high concentrations of C-protein molecules may act in a hitherto novel mechanism on PKR to inhibit host cell protein synthesis during viral infection.     

Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation

Alphavirus infection results in the shutoff of host protein synthesis in favor of viral translation. Here, we show that during Semliki Forest virus (SFV) infection, the translation inhibition is largely due to the activation of the cellular stress response via phosphorylation of eukaryotic translation initiation factor 2alpha subunit (eIF2alpha). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2alpha does not result in efficient shutoff, despite efficient viral protein production. Furthermore, we show that the SFV translation enhancer element counteracts the translation inhibition imposed by eIF2alpha phosphorylation. In wild-type MEFs, viral infection induces the transient formation of stress granules (SGs) containing the cellular TIA-1/R proteins. These SGs are disassembled in the vicinity of viral RNA replication, synchronously with the switch from cellular to viral gene expression. We propose that phosphorylation of eIF2alpha and the consequent SG assembly is important for shutoff to occur and that the localized SG disassembly and the presence of the enhancer aid the SFV mRNAs to elude general translational arrest.     

Rubella virus capsid protein interacts with poly(a)-binding protein and inhibits translation

During virus assembly, the capsid proteins of RNA viruses bind to genomic RNA to form nucleocapsids. However, it is now evident that capsid proteins have additional functions that are unrelated to nucleocapsid formation. Specifically, their interactions with cellular proteins may influence signaling pathways or other events that affect virus replication. Here we report that the rubella virus (RV) capsid protein binds to poly(A)-binding protein (PABP), a host cell protein that enhances translational efficiency by circularizing mRNAs. Infection of cells with RV resulted in marked increases in the levels of PABP, much of which colocalized with capsid in the cytoplasm. Mapping studies revealed that capsid binds to the C-terminal half of PABP, which interestingly is the region that interacts with other translation regulators, including PABP-interacting protein 1 (Paip1) and Paip2. The addition of capsid to in vitro translation reaction mixtures inhibited protein synthesis in a dose-dependent manner; however, the capsid block was alleviated by excess PABP, indicating that inhibition of translation occurs through a stoichiometric mechanism. To our knowledge, this is the first report of a viral protein that inhibits protein translation by sequestration of PABP. We hypothesize that capsid-dependent inhibition of translation may facilitate the switch from viral translation to packaging RNA into nucleocapsids.     

Expression of Semliki Forest virus capsid protein from SV40 recombinant virus

Here, the proteolytic processing of the Semliki Forest virus (SFV) capsid protein was studied in the absence of other viral functions. Two different fragments of the SFV messenger cDNA, coding for capsid protein and 174 and 38 extra amino acids from the envelope proteins, respectively, were cloned in the late region of the SV40 viral DNA. Cells infected with the SV40 recombinant virus stocks were analyzed for the production of SFV capsid mRNA and polypeptide. Immunofluorescence staining of the infected cells indicated that the produced SFV capsid protein accumulated mainly in the nucleus. Polyacrylamide gel electrophoresis of the immunoprecipitated SFV capsid proteins showed that both recombinants yielded a labelled band equivalent in size to the SFV capsid protein. Thus the proteolytic processing takes place even under conditions where the capsid protein is the only virus-specified protein synthesized.     

Viral alteration of cellular translational machinery increases defective ribosomal products

Here we show that cells expressing genes inserted into Semliki Forest virus (SFV) vectors generate a large fraction of defective ribosomal products (DRiPs) due to frequent initiation on downstream Met residues. In monopolizing the host cell translational machinery, SFV reduces levels of translation eukaryotic initiation factor 4E (eIF4E), diminishes phosphorylation of ribosome subunit S6, and phosphorylates translation initiation factor eIF2alpha. We show that the last event is required for SFV mistranslation of inserted genes. Downstream initiation is suppressed by fusing inserted genes with the open reading frame encoding the SFV capsid, demonstrating that one function of the capsid element is to enable ribosomes to initiate translation in the proper location. These results show that in modifying translation, viral vectors can unpredictably increase the generation of truncated polypeptides and thereby the DRiP fraction of inserted gene products, which can potentially affect their yield, therapeutic efficacy, and immunogenicity.     

Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages

Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.     

Alphaviruses as Tools in Neurobiology and Gene Therapy

Abstract

The broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80–95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing β-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells.     

Alphaviruses as tools in neurobiology and gene therapy

The broad host range and superior infectivity of alphaviruses have encouraged the development of efficient expression vectors for Semliki Forest virus (SFV) and Sindbis virus (SIN). The generation of high-titer recombinant alphavirus stocks has allowed high-level expression of a multitude of nuclear, cytoplasmic, membrane-associated and secreted proteins in a variety of different cell lines and primary cell cultures. Despite the viral cytopathogenic effects, functional assays on recombinant proteins are possible for a time-period of at least 24 hours post-infection. The high percentage (80-95%) of primary neurons infected with SFV has allowed localization and functional studies of recombinant proteins in these primary cell cultures. Through multiple infection studies the interaction of receptor and G protein subunits has become feasible. Establishment of efficient scale-up procedures has allowed production of large quantities of recombinant protein. Potential gene therapy applications of alphaviruses could be demonstrated by injection of recombinant SIN particles expressing beta-galactosidase into mouse brain. Tissue/cell specific infection has been achieved by introduction of an IgG-binding domain of protein A domain into one of the spike proteins of SIN. This enabled efficient targeting of infection to human lymphoblastoid cells.     

Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.     

RNA interference-mediated inhibition of Semliki Forest virus replication in mammalian cells

RNA interference (RNAi) has recently shown promise as a mode of inhibition of slowly replicating viruses causing chronic diseases such as hepatitis C. To investigate whether RNAi is also feasible for rapidly growing RNA viruses such as alphaviruses, we tested the ability of expressed short hairpin RNAs (shRNAs) to inhibit the Semliki Forest virus (SFV), a rapidly replicating positive-strand RNA virus. Plasmids expressing shRNAs targeting SFV target sequences under the control of a human U6 promoter were introduced into BHK-21 cells. The targets included sequences encoding nonstructural (nsP1, 2, and 4) and structural (capsid) proteins as well as nonviral sequences serving as control targets. Twenty-four to 48 hours following transfection with shRNA plasmids, the cells were infected with replication-competent or replication-deficient recombinant SFV expressing green fluorescent protein (GFP) at a multiplicity of infection (MOI) of approximately 5. Viral replication was monitored by fluorescence microscopy and flow cytometry. Specific and marked reduction of viral replication was observed with shRNAs targeting nsP1 and nsP4. The degree of inhibition of the replication-deficient SFV was >or=70% over a 5-day period, a level similar to the transfection efficiency, suggesting complete inhibition of nonreplicating virus in the transfected cell population. However, only nsP1 shRNA was inhibitory against replication-competent SFV (approximately 30%-50% reduction), and this effect was transient. No inhibition was observed with control shRNAs. In contrast to the recent success of RNAi approaches for slowly growing viruses, these results illustrate the challenge of inhibiting very rapidly replicating RNA viruses by RNAi. However, the addition of RNAi approaches to other antiviral modalities might improve the response to acute infections.     

Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification

Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.     

M-X-I motif of semliki forest virus capsid protein affects nucleocapsid assembly

Alphavirus budding is driven by interactions between spike and nucleocapsid proteins at the plasma membrane. The binding motif, Y-X-L, on the spike protein E2 and the corresponding hydrophobic cavity on the capsid protein were described earlier. The spike-binding cavity has also been suggested to bind an internal hydrophobic motif, M113-X-I115, of the capsid protein. In this study we found that replacement of amino acids M113 and I115 with alanines, as single or double mutations, abolished formation of intracellular nucleocapsids. The mutants could still bud efficiently, but the NCs in the released virions were not stable after removal of the membrane and spike protein layer. In addition to wild-type spherical particles, elongated multicored particles were found at the plasma membrane and released from the host cell. We conclude that the internal capsid motif has a biological function in the viral life cycle, especially in assembly of nucleocapsids. We also provide further evidence that alphaviruses may assemble and bud from the plasma membrane in the absence of preformed nucleocapsids.     

The kinase inhibitor SFV785 dislocates dengue virus envelope protein from the replication complex and blocks virus assembly

Dengue virus (DENV) is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785), which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.     

Adenovirus inhibition of cellular protein synthesis involves inactivation of cap-binding protein.

Adenovirus (Ad) infection results in a marked inhibition of cellular protein synthesis that initiates during the late phase of the viral infectious cycle. We show that the mechanism used for suppression of cellular protein synthesis during cell cycle progression is exploited by Ad to repress host and enhance late viral mRNA translation. Discrimination between cellular and late Ad mRNAs and inhibition of host protein synthesis are shown to involve viral-mediated underphosphorylation of cap-binding protein (CBP) and subsequent inactivation of CBP complex, a large enzymatic complex required for cap-dependent mRNA translation. Late Ad mRNAs, like those of poliovirus, possess the unique ability to translate independent of a normal cap recognition process and do not require the activity of CBP complex. Inhibition of cellular translation by these two viruses is quite similar, except that whereas CBP complex is proteolytically degraded by poliovirus, it is functionally inactivated by Ad.     

Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study.

According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus polyprotein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracellular form of the viral capsid protein (Cint) retaining the prM signal sequence at its carboxy terminus. This hydrophobic anchor is subsequently removed by the viral protease, resulting in formation of the mature viral capsid protein found in virions (Cvir). We have prepared in vitro expression cassettes coding for both forms of the capsid protein, for the prM protein, for the C-prM precursor, and for the viral protease components of West Nile flavivirus and characterized their translation products. Using Cint and Cvir translation products as molecular markers, we have observed processing of the intracellular form of the West Nile capsid protein by the viral protease in vitro both upon cotranslation of the C-prM precursor and the viral protease-encoding cassette and by incubation of C-prM translation products with a detergent-solubilized extract of cells infected with a recombinant vaccinia virus expressing the active viral protease. The cleavage of Cint by the viral protease at the predicted dibasic site was verified by introduction of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease.     

Rapamycin and Wortmannin Enhance Replication of a Defective Encephalomyocarditis Virus

Inhibitors of the phosphatidylinositol 3-kinase (PI3 kinase)–FKBP-rapamycin-associated protein (FRAP) pathway, such as rapamycin and wortmannin, induce dephosphorylation and activation of the suppressor of cap-dependent translation, 4E-BP1. Encephalomyocarditis virus (EMCV) infection leads to activation of 4E-BP1 at the time of host translation shutoff. Consistent with these data, rapamycin mildly enhances the synthesis of viral proteins and the shutoff of host cell protein synthesis after EMCV infection. In this study, two defective EMCV strains were generated by deleting portions of the 2A coding region of an infectious cDNA clone. These deletions dramatically decreased the efficiency of viral protein synthesis and abolished the virus-induced shutoff of host translation after infection of BHK-21 cells. Both translation and processing of the P1-2A capsid precursor polypeptide are impaired by the deletions in 2A. The translation and yield of mutant viruses were increased significantly by the presence of rapamycin and wortmannin during infection. Thus, inhibition of the PI3 kinase-FRAP signaling pathway partly complements mutations in 2A protein and reverses a slow-virus phenotype.     

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.     

Viral and host proteins involved in picornavirus life cycle

Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions.