Human NLRP1 is a sensor for double-stranded RNA.

Inflammasomes function as intracellular sensors of pathogen infection or cellular perturbation and thereby play a central role in numerous diseases. Given the high abundance of NLRP1 in epithelial barrier tissues, we screened a diverse panel of viruses for inflammasome activation in keratinocytes. We identified Semliki Forest virus (SFV), a positive-strand RNA virus, as a potent activator of human but not murine NLRP1B. SFV replication and the associated formation of double-stranded (ds) RNA was required to engage the NLRP1 inflammasome. Moreover, delivery of long dsRNA was sufficient to trigger activation. Biochemical studies revealed that NLRP1 binds dsRNA through its leucine-rich repeat domain, resulting in its NACHT domain gaining adenosine triphosphatase activity. Altogether, these results establish human NLRP1 as a direct sensor for dsRNA and thus RNA virus infection.

Characterization of virus-mediated immunogenic cancer cell death and the consequences for oncolytic virus-based immunotherapy of cancer.

Oncolytic viruses have the potential to induce immunogenic cell death (ICD) that may provoke potent and long-lasting anti-cancer immunity. Here we aimed to characterize the ICD-inducing ability of wild-type Adenovirus (Ad), Semliki Forest virus (SFV) and Vaccinia virus (VV). We did so by investigating the cell death and immune-activating properties of virus-killed tumor cells. Ad-infection of tumor cells primarily activates autophagy, but also activate events of necroptotic and pyroptotic cell death. SFV infection on the other hand primarily activates immunogenic apoptosis while VV activates necroptosis. All viruses mediated lysis of tumor cells leading to the release of danger-associated molecular patterns, triggering of phagocytosis and maturation of dendritic cells (DCs). However, only SFV-infected tumor cells triggered significant T helper type 1 (Th1)-cytokine release by DCs and induced antigen-specific T-cell activation. Our results elucidate cell death processes activated upon Ad, SFV, and VV infection and their potential to induce T cell-mediated anti-tumor immune responses. This knowledge provides important insight for the choice and design of therapeutically successful virus-based immunotherapies.

Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery.

G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP. To study potential proviral roles of G3BP, we used human osteosarcoma (U2OS) cell lines lacking endogenous G3BP generated using CRISPR-Cas9 and reconstituted with a panel of G3BP1 mutants and truncation variants. While SFV replicated with varying efficiency in all cell lines, CHIKV could only replicate in cells expressing G3BP1 variants containing both the NTF2-like and the RGG domains. The ability of SFV to replicate in the absence of G3BP allowed us to study effects of different domains of the protein. We used immunoprecipitation to demonstrate that that both NTF2-like and RGG domains are necessary for the formation a complex between nsP3, G3BP1 and the 40S ribosomal subunit. Electron microscopy of SFV-infected cells revealed that formation of nsP3:G3BP1 complexes via the NTF2-like domain was necessary for clustering of cytopathic vacuoles (CPVs) and that the presence of the RGG domain was necessary for accumulation of electron dense material containing G3BP1 and nsP3 surrounding the CPV clusters. Clustered CPVs also exhibited localised high levels of translation of viral mRNAs as detected by ribopuromycylation staining. These data confirm that G3BP is a ribosomal binding protein and reveal that alphaviral nsP3 uses G3BP to concentrate viral replication complexes and to recruit the translation initiation machinery, promoting the efficient translation of viral mRNAs.

Timeliness of Proteolytic Events Is Prerequisite for Efficient Functioning of the Alphaviral Replicase.

Polyprotein processing has an important regulatory role in the life cycle of positive-strand RNA viruses. In the case of alphaviruses, sequential cleavage of the nonstructural polyprotein (ns-polyprotein) at three sites eventually yields four mature nonstructural proteins (nsPs) that continue working in complex to replicate viral genomic RNA and transcribe subgenomic RNA. Recognition of cleavage sites by viral nsP2 protease is guided by short sequences upstream of the scissile bond and, more importantly, by the spatial organization of the replication complex. In this study, we analyzed the consequences of the artificially accelerated processing of the Semliki Forest virus ns-polyprotein. It was found that in mammalian cells, not only the order but also the correct timing of the cleavage events is essential for the success of viral replication. Analysis of the effects of compensatory mutations in rescued viruses as well as in vitro translation and trans-replicase assays corroborated our findings and revealed the importance of the V515 residue in nsP2 for recognizing the P4 position in the nsP1/nsP2 cleavage site. We also extended our conclusions to Sindbis virus by analyzing the properties of the hyperprocessive variant carrying the N614D mutation in nsP2. We conclude that the sequence of the nsP1/nsP2 site in alphaviruses is under selective pressure to avoid the presence of sequences that are recognized too efficiently and would otherwise lead to premature cleavage at this site before completion of essential tasks of RNA synthesis or virus-induced replication complex formation. Even subtle changes in the ns-polyprotein processing pattern appear to lead to virus attenuation.IMPORTANCE The polyprotein expression strategy is a cornerstone of alphavirus replication. Three sites within the ns-polyprotein are recognized by the viral nsP2 protease and cleaved in a defined order. Specific substrate targeting is achieved by the recognition of the short sequence upstream of the scissile bond and a correct macromolecular assembly of ns-polyprotein. Here, we highlighted the importance of the timeliness of proteolytic events, as an additional layer of regulation of efficient virus replication. We conclude that, somewhat counterintuitively, the cleavage site sequences at the nsP1/nsP2 and nsP2/nsP3 junctions are evolutionarily selected to be recognized by protease inefficiently, to avoid premature cleavages that would be detrimental for the assembly and functionality of the replication complex. Understanding the causes and consequences of viral polyprotein processing events is important for predicting the properties of mutant viruses and should be helpful for the development of better vaccine candidates and understanding potential mechanisms of resistance to protease inhibitors.

Alphavirus-induced hyperactivation of PI3K/AKT directs pro-viral metabolic changes.

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease.

An Alphavirus E2 Membrane-Proximal Domain Promotes Envelope Protein Lateral Interactions and Virus Budding.

Alphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. The virus membrane is covered by a lattice composed of 80 spikes, each a trimer of heterodimers of the E2 and E1 transmembrane proteins. During virus endocytic entry, the E1 glycoprotein mediates the low-pH-dependent fusion of the virus membrane with the endosome membrane, thus initiating virus infection. While much is known about E1 structural rearrangements during membrane fusion, it is unclear how the E1/E2 dimer dissociates, a step required for the fusion reaction. A recent Alphavirus cryo-electron microscopy reconstruction revealed a previously unidentified D subdomain in the E2 ectodomain, close to the virus membrane. A loop within this region, here referred to as the D-loop, contains two highly conserved histidines, H348 and H352, which were hypothesized to play a role in dimer dissociation. We generated Semliki Forest virus mutants containing the single and double alanine substitutions H348A, H352A, and H348/352A. The three D-loop mutations caused a reduction in virus growth ranging from 1.6 to 2 log but did not significantly affect structural protein biosynthesis or transport, dimer stability, virus fusion, or specific infectivity. Instead, growth reduction was due to inhibition of a late stage of virus assembly at the plasma membrane. The virus particles that are produced show reduced thermostability compared to the wild type. We propose the E2 D-loop as a key region in establishing the E1-E2 contacts that drive glycoprotein lattice formation and promote Alphavirus budding from the plasma membrane.IMPORTANCEAlphavirus infection causes severe and debilitating human diseases for which there are no effective antiviral therapies or vaccines. In order to develop targeted therapeutics, detailed molecular understanding of the viral entry and exit mechanisms is required. In this report, we define the role of the E2 protein juxtamembrane D-loop, which contains highly conserved histidine residues at positions 348 and 352. These histidines do not play an important role in virus fusion and infection. However, mutation of the D-loop histidines causes significant decreases in the assembly and thermostability of Alphavirus particles. Our results suggest that the E2 D-loop interacts with the E1 protein to promote Alphavirus budding.

Immunogenicity of virus-like Semliki Forest virus replicon particles expressing Indian HIV-1C gag, env and polRT genes.

Development of a vaccine targeting human immunodeficiency virus-1 subtype C (HIV-1C) is an important public health priority in regions with a high prevalence of the clade C virus. The present study demonstrates the immunogenicity of recombinant Semliki Forest virus (SFV)-based virus-like replicon particles (VRPs) expressing Indian HIV-1C env/gag/polRT genes. Immunization of mice with recombinant VRPs in a homologous prime-boost protocol, either individually or in combination, elicited significant antigen-specific IFN-γ T cell responses as detected by the ELISPOT assay. Additionally, Gag-specific TNF-α secreting CD8+ and CD4+ T cells and Env-specific IL-2 secreting T cells were also elicited by mice immunized with Gag and Env constructs, respectively, as estimated by intracellular cytokine staining assay. Moreover, an HIV Pol-specific TNF-α response was elicited in mice immunized with a combination of the three VRP constructs. Furthermore, HIV-1C Gag and Env-specific binding antibodies were elicited as verified by gp120 ELISA and p24 Gag ELISA, respectively. The immunogenicity of VRPs was found to be higher as compared to that of RNA replicons and VRPs may therefore be promising preventive and therapeutic candidate vaccines for the control and management of HIV/AIDS.

Alphavirus Restriction by IFITM Proteins.

Interferon inducible transmembrane proteins (IFITMs) are broad-spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo-, flavi-, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid-induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH-induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV.

MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma.

UNLABELLED: Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem that severely restricts viral replication and oncolysis. We show here that the Semliki Forest virus (SFV) strain SFV4, which causes lethal encephalitis in mice, is able to infect and replicate independent of the IFN-I defense in mouse glioblastoma cells and cell lines originating from primary human glioblastoma patient samples. The ability to tolerate IFN-I was retained in SFV4-miRT124 cells, a derivative cell line of strain SFV4 with a restricted capacity to replicate in neurons due to insertion of target sites for neuronal microRNA 124. The IFN-I tolerance was associated with the viral nsp3-nsp4 gene region and distinct from the genetic loci responsible for SFV neurovirulence. In contrast to the naturally attenuated strain SFV A7(74) and its derivatives, SFV4-miRT124 displayed increased oncolytic potency in CT-2A murine astrocytoma cells and in the human glioblastoma cell lines pretreated with IFN-I. Following a single intraperitoneal injection of SFV4-miRT124 into C57BL/6 mice bearing CT-2A orthotopic gliomas, the virus homed to the brain and was amplified in the tumor, resulting in significant tumor growth inhibition and improved survival. IMPORTANCE: Although progress has been made in development of replicative oncolytic viruses, information regarding their overall therapeutic potency in a clinical setting is still lacking. This could be at least partially dependent on the IFN-I sensitivity of the viruses used. Here, we show that the conditionally replicating SFV4-miRT124 virus shares the IFN-I tolerance of the pathogenic wild-type SFV, thereby allowing efficient targeting of a glioma that is refractory to naturally attenuated therapy vector strains sensitive to IFN-I. This is the first evidence of orthotopic syngeneic mouse glioma eradication following peripheral alphavirus administration. Our findings indicate a clear benefit in harnessing the wild-type virus replicative potency in development of next-generation oncolytic alphaviruses.

A novel mitochondrial MAVS/Caspase-8 platform links RNA virus-induced innate antiviral signaling to Bax/Bak-independent apoptosis.

Semliki Forest virus (SFV) requires RNA replication and Bax/Bak for efficient apoptosis induction. However, cells lacking Bax/Bak continue to die in a caspase-dependent manner. In this study, we show in both mouse and human cells that this Bax/Bak-independent pathway involves dsRNA-induced innate immune signaling via mitochondrial antiviral signaling (MAVS) and caspase-8. Bax/Bak-deficient or Bcl-2- or Bcl-xL-overexpressing cells lacking MAVS or caspase-8 expression are resistant to SFV-induced apoptosis. The signaling pathway triggered by SFV does neither involve death receptors nor the classical MAVS effectors TNFR-associated factor-2, IRF-3/7, or IFN-ß but the physical interaction of MAVS with caspase-8 on mitochondria in a FADD-independent manner. Consistently, caspase-8 and -3 activation are reduced in MAVS-deficient cells. Thus, after RNA virus infection MAVS does not only elicit a type I antiviral response but also recruits caspase-8 to mitochondria to mediate caspase-3 activation and apoptosis in a Bax/Bak-independent manner.

Therapeutic immunization and local low-dose tumor irradiation, a reinforcing combination.

Therapeutic cancer vaccines show promise in preclinical studies, yet their clinical efficacy is limited. Increased recruitment of immune cells into tumors and suppression of the immune suppressive tumor environment are critical components toward effective cancer immunotherapies. Here, we report how local low-dose irradiation, alone or with a therapeutic immunization based on Semliki Forest virus (SFV) against human papillomavirus (HPV)-related cancer, influences these immune mechanisms. We first demonstrated that immunization with SFVeE6,7 or SFVeOVA, replicon particles expressing either HPV16 E6/E7 or ovalbumin, resulted in an antigen-specific migration of CD8+ T cells into HPV- and OVA-specific tumors. Local low-dose tumor irradiation alone resulted in a 2-fold increase of intratumoral CD8+ T cells. When 14 Gy irradiation was combined with immunization, intratumoral numbers of CD8+ T cells increased 10-fold and the number of CD8+ T cells specific for the E7- epitope increased more than 20-fold. Irradiation alone however also increased the number of intratumoral myeloid-derived suppressor cells (MDSCs) 3.5-fold. Importantly, this number did not further increase when combined with immunization. As a result, the ratio of antigen-specific CD8+ T cells and MDSCs in tumors increased up to 85-fold compared to the control. We furthermore demonstrated that following irradiation CCR2 and CCL2, CXCR6 and CCL16, chemokines and ligands involved in tumor homing of immune cells, were significantly up regulated. This study demonstrates that local low-dose tumor irradiation influences the intratumoral immune population induced by SFVeE6,7 immunization by a strong increase in the ratio of antitumoral to immune suppressive cells, thus changing the intratumoral immune balance in favor of antitumor activity.

Control of the rescue and replication of Semliki Forest virus recombinants by the insertion of miRNA target sequences.

Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3' untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability.

RIG-I and MDA-5 detection of viral RNA-dependent RNA polymerase activity restricts positive-strand RNA virus replication.

Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5'-triphosphate (5'-ppp) RNA and mediate IFN production. Cytosolic 5'-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-ß independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5'-ppp dsRNA and induces IFN-ß through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-ß induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-ß production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.

The role of E3 in pH protection during alphavirus assembly and exit.

Alphaviruses are small enveloped viruses whose surface is covered by spikes composed of trimers of E2/E1 glycoprotein heterodimers. During virus entry, the E2/E1 dimer dissociates within the acidic endosomal environment, freeing the E1 protein to mediate fusion of the viral and endosome membranes. E2 is synthesized as a precursor, p62, which is cleaved by furin in the late secretory pathway to produce mature E2 and a small peripheral glycoprotein, E3. The immature p62/E1 dimer is acid resistant, but since p62 is cleaved before exit from the acidic secretory pathway, low pH-dependent binding of E3 to the spike complex is believed to prevent premature fusion. Based on analysis of the structure of the Chikungunya virus E3/E2/E1 complex, we hypothesized that interactions of E3 residues Y47 and Y48 with E2 are important in this binding. We then directly tested the in vivo role of E3 in pH protection by alanine substitutions of E3 Y47 and Y48 (Y47/48A) in Semliki Forest virus. The mutant was nonviable and was blocked in E1 transport to the plasma membrane and virus production. Although the Y47/48A mutant initially formed the p62/E1 heterodimer, the dimer dissociated during transport through the secretory pathway. Neutralization of the pH in the secretory pathway successfully rescued dimer association, E1 transport, and infectious particle production. Further mutagenesis identified the critical contact as the cation-π interaction of E3 Y47 with E2. Thus, E3 mediates pH protection of E1 during virus biogenesis via interactions strongly dependent on Y47 at the E3-E2 interface.

Magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of Semliki Forest virus.

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection.

Cross-inhibition of chikungunya virus fusion and infection by alphavirus E1 domain III proteins.

Alphaviruses are small enveloped RNA viruses that include important emerging human pathogens, such as chikungunya virus (CHIKV). These viruses infect cells via a low-pH-triggered membrane fusion reaction, making this step a potential target for antiviral therapies. The E1 fusion protein inserts into the target membrane, trimerizes, and refolds to a hairpin-like conformation in which the combination of E1 domain III (DIII) and the stem region (DIII-stem) pack against a core trimer composed of E1 domains I and II (DI/II). Addition of exogenous DIII proteins from Semliki Forest virus (SFV) has been shown to inhibit E1 hairpin formation and SFV fusion and infection. Here we produced and characterized DIII and DI/II proteins from CHIKV and SFV. Unlike SFV DIII, both core trimer binding and fusion inhibition by CHIKV DIII required the stem region. CHIKV DIII-stem and SFV DIII-stem showed efficient cross-inhibition of SFV, Sindbis virus, and CHIKV infections. We developed a fluorescence anisotropy-based assay for the binding of SFV DIII-stem to the core trimer and used it to demonstrate the relatively high affinity of this interaction (Kd [dissociation constant], ∼85 nM) and the importance of the stem region. Together, our results support the conserved nature of the key contacts of DIII-stem in the alphavirus E1 homotrimer and describe a sensitive and quantitative in vitro assay for this step in fusion protein refolding.

Attenuation of Semliki Forest virus neurovirulence by microRNA-mediated detargeting.

Artificial target sequences for tissue-specific miRNAs have recently been introduced as a new means for altering the tissue tropism of viral replication. This approach can be used to improve the safety of oncolytic viruses for cancer virotherapy by restricting their replication in unwanted tissues, such as the liver. Semliki Forest virus (SFV) is a positive-strand RNA virus and, similar to the related alphaviruses, like Sindbis virus, has potential as a gene therapy vector and an oncolytic virotherapy agent, but this potential is limited by the neurovirulence of these alphaviruses. Here, we have generated a replicative SFV4 carrying six tandem targets for the neuron-specific miR124 between the viral nonstructural protein 3 and 4 (nsp3 and nsp4) genes. When administered intraperitoneally into adult BALB/c mice, SFV4-miRT124 displayed an attenuated spread into the central nervous system (CNS) and greatly increased survival. Peripheral replication was not affected, indicating neuron-specific attenuation. Moreover, a strong protective SFV immunity was elicited in these animals. Intracranial infection of adult mice with SFV4-miRT124 showed greatly reduced infection of neurons in the brain but led to the infection of oligodendrocytes in the corpus callosum. Taken together, our data show that miR124-mediated attenuation of neurovirulence is a feasible and promising strategy for generating safer oncolytic alphavirus virotherapy agents.

Accumulation of autophagosomes in Semliki Forest virus-infected cells is dependent on expression of the viral glycoproteins.

Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH(4)Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH(4)Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.

Identification of a specific region in the e1 fusion protein involved in zinc inhibition of semliki forest virus fusion.

The enveloped alphaviruses infect cells via a low-pH-triggered membrane fusion reaction mediated by the viral transmembrane protein E1. During fusion, E1 inserts into the target membrane and refolds to a hairpin-like postfusion conformation in which domain III (DIII) and the juxtamembrane stem pack against a central core trimer. Although zinc has previously been shown to cause a striking block in alphavirus fusion with liposome target membranes, the mechanism of zinc's effect on the E1 fusion protein is not understood. Here we developed a cell culture system to study zinc inhibition of fusion and infection of the alphavirus Semliki Forest virus (SFV). Inclusion of 2 mM ZnCl(2) in the pH 5.75 fusion buffer caused a decrease of ∼5 logs in SFV fusion at the plasma membrane. Fusion was also inhibited by nickel, a chemically related transition metal. Selection for SFV zinc resistance identified a key histidine residue, H333 on E1 DIII, while other conserved E1 histidine residues were not involved. An H333N mutation conferred resistance to both zinc and nickel, with properties in keeping with the known pH-dependent chelation of these metals by histidine. Biochemical studies demonstrated that zinc strongly inhibits formation of the postfusion E1 trimer in wild-type SFV but not in an H333 mutant. Together our results suggest that zinc acts by blocking the fold-back of DIII via its interaction with H333.

Macromolecular assembly-driven processing of the 2/3 cleavage site in the alphavirus replicase polyprotein.

Semliki Forest virus (SFV) is a member of the Alphavirus genus, which produces its replicase proteins in the form of a nonstructural (ns) polyprotein precursor P1234. The maturation of the replicase occurs in a temporally controlled manner by protease activity of nsP2. The template preference and enzymatic capabilities of the alphaviral replication complex have a very important connection with its composition, which is irreversibly altered by proteolysis. The final cleavage of the 2/3 site in the ns polyprotein apparently leads to significant rearrangements within the replication complex and thus denotes the "point of no return" for viral replication progression. Numerous studies have devised rules for when and how ns protease acts, but how the alphaviral 2/3 site is recognized remained largely unexplained. In contrast to the other two cleavage sites within the ns polyprotein, the 2/3 site evidently lacks primary sequence elements in the vicinity of the scissile bond sufficient for specific protease recognition. In this study, we sought to investigate the molecular details of the regulation of the 2/3 site processing in the SFV ns polyprotein. We present evidence that correct macromolecular assembly, presumably strengthened by exosite interactions rather than the functionality of the individual nsP2 protease, is the driving force for specific substrate targeting. We conclude that structural elements within the macrodomain of nsP3 are used for precise positioning of a substrate recognition sequence at the catalytic center of the protease and that this process is coordinated by the exact N-terminal end of nsP2, thus representing a unique regulation mechanism used by alphaviruses.