Getah virus capsid protein undergoes co-condensation with viral genomic RNA to facilitate virion assembly.

Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.

Autoinhibition of suicidal capsid protease from O'nyong'nyong virus.

Alphaviruses pose a significant threat to public health. Capsid protein encoded in the alphaviral genomes constitutes an interesting therapy target, as it also serves as a protease (CP). Remarkably, it undergoes autoproteolysis, leading to the generation of the C-terminal tryptophan that localizes to the active pocket, deactivating the enzyme. Lack of activity hampers the viral replication cycle, as the virus is not capable of producing the infectious progeny. We investigated the structure and function of the CP encoded in the genome of O'nyong'nyong virus (ONNV), which has instigated outbreaks in Africa. Our research provides a high-resolution crystal structure of the ONNV CP in its active state and evaluates the enzyme's activity. Furthermore, we demonstrated a dose-dependent reduction in ONNV CP proteolytic activity when exposed to indole, suggesting that tryptophan analogs may be a promising basis for developing small molecule inhibitors. It's noteworthy that the capsid protease plays an essential role in virus assembly, binding viral glycoproteins through its glycoprotein-binding hydrophobic pocket. We showed that non-aromatic cyclic compounds like dioxane disrupt this vital interaction. Our findings provide deeper insights into ONNV's biology, and we believe they will prove instrumental in guiding the development of antiviral strategies against arthritogenic alphaviruses.

The many ways in which alphaviruses bind to cells.

Only a subset of viruses can productively infect many different host species. Some arthropod-transmitted viruses, such as alphaviruses, can infect invertebrate and vertebrate species including insects, reptiles, birds, and mammals. This broad tropism may be explained by their ability to engage receptors that are conserved across vertebrate and invertebrate classes. Through several genome-wide loss-of-function screens, new alphavirus receptors have been identified, some of which bind to multiple related viruses in different antigenic complexes. Structural analysis has revealed that distinct sites on the alphavirus glycoprotein can mediate receptor binding, which opposes the idea that a single receptor-binding site mediates viral entry. Here, we discuss how different paradigms of receptor engagement on cells might explain the promiscuity of alphaviruses for multiple hosts.

The roles of 6K protein on Getah virus replication and pathogenicity.

Alphavirus is a type of arbovirus that can infect both humans and animals. The amino acid sequence of the 6K protein, being one of the structural proteins of the alphavirus, is not conserved. Deletion of this protein will result in varying effects on different alphaviruses. Our study focuses on the function of the Getah virus (GETV) 6K protein in infected cells and mice. We successfully constructed infectious clone plasmids and created resulting viruses (rGETV and rGETV-Δ6K). Our comprehensive microscopic analysis revealed that the 6 K protein mainly stays in the endoplasmic reticulum. In addition, rGETV-Δ6K has lower thermal stability and sensitivity to temperature than GETV. Although the deletion of the 6K protein does not reduce virion production in ST cells, it affects the release of virions from host cells by inhibiting the process of E2 protein transportation to the plasma membrane. Subsequent in vivo testing demonstrated that neonatal mice infected with rGETV-Δ6K had a lower virus content, less significant pathological changes in tissue slices, and milder disease than those infected with the wild-type virus. Our results indicate that the 6K protein effectively reduces the viral titer by influencing the release of viral particles. Furthermore, the 6K protein play a role in the clinical manifestation of GETV disease.

Human Synovial Mesenchymal Stem Cells Expressed Immunoregulatory Factors IDO and TSG6 in a Context of Arthritis Mediated by Alphaviruses.

Infection by arthritogenic alphaviruses (aavs) can lead to reactive arthritis, which is characterized by inflammation and persistence of the virus; however, its mechanisms remain ill-characterized. Intriguingly, it has been shown that viral persistence still takes place in spite of robust innate and adaptive immune responses, characterized notably by the infiltration of macrophages (sources of TNF-alpha) as well as T/NK cells (sources of IFN-gamma) in the infected joint. Aavs are known to target mesenchymal stem cells (MSCs) in the synovium, and we herein tested the hypothesis that the infection of MSCs may promote the expression of immunoregulators to skew the anti-viral cellular immune responses. We compared the regulated expression via human synovial MSCs of pro-inflammatory mediators (e.g., IL-1ß, IL6, CCL2, miR-221-3p) to that of immunoregulators (e.g., IDO, TSG6, GAS6, miR146a-5p). We used human synovial tissue-derived MSCs which were infected with O'Nyong-Nyong alphavirus (ONNV, class II aav) alone, or combined with recombinant human TNF-α or IFN-γ, to mimic the clinical settings. We confirmed via qPCR and immunofluorescence that ONNV infected human synovial tissue-derived MSCs. Interestingly, ONNV alone did not regulate the expression of pro-inflammatory mediators. In contrast, IDO, TSG6, and GAS6 mRNA expression were increased in response to ONNV infection alone, but particularly when combined with both recombinant cytokines. ONNV infection equally decreased miR-146a-5p and miR-221-3p in the untreated cells and abrogated the stimulatory activity of the recombinant TNF-α but not the IFN-gamma. Our study argues for a major immunoregulatory phenotype of MSCs infected with ONNV which may favor virus persistence in the inflamed joint.

Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression.

IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

Alphavirus Evasion of Zinc Finger Antiviral Protein (ZAP) Correlates with CpG Suppression in a Specific Viral nsP2 Gene Sequence.

Certain re-emerging alphaviruses, such as chikungunya virus (CHIKV), cause serious disease and widespread epidemics. To develop virus-specific therapies, it is critical to understand the determinants of alphavirus pathogenesis and virulence. One major determinant is viral evasion of the host interferon response, which upregulates antiviral effectors, including zinc finger antiviral protein (ZAP). Here, we demonstrated that Old World alphaviruses show differential sensitivity to endogenous ZAP in 293T cells: Ross River virus (RRV) and Sindbis virus (SINV) are more sensitive to ZAP than o'nyong'nyong virus (ONNV) and CHIKV. We hypothesized that the more ZAP-resistant alphaviruses evade ZAP binding to their RNA. However, we did not find a correlation between ZAP sensitivity and binding to alphavirus genomic RNA. Using a chimeric virus, we found the ZAP sensitivity determinant lies mainly within the alphavirus non-structural protein (nsP) gene region. Surprisingly, we also did not find a correlation between alphavirus ZAP sensitivity and binding to nsP RNA, suggesting ZAP targeting of specific regions in the nsP RNA. Since ZAP can preferentially bind CpG dinucleotides in viral RNA, we identified three 500-bp sequences in the nsP region where CpG content correlates with ZAP sensitivity. Interestingly, ZAP binding to one of these sequences in the nsP2 gene correlated to sensitivity, and we confirmed that this binding is CpG-dependent. Our results demonstrate a potential strategy of alphavirus virulence by localized CpG suppression to evade ZAP recognition.

Structure of Semliki Forest virus in complex with its receptor VLDLR.

Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378 Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions at multiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV.

The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

Interaction models between peptide substrate and Alphavirus Protease nsP2 of Chikungunya and Mayaro and implications to the mechanism of action.

The Arbovirus (Arthropod-borne virus) is a group which comprises viruses whose transmission is carried out by arthropod vectors infecting vertebrates. Some arboviruses related to human diseases have been given considerable relevance as Chikungunya and Mayaro of the family Togaviridae, genus Alphavirus. The lack of proper specific treatment has prompted the requirement for deeper structural studies that could unveil leads to new drugs. Among possible targets, viral proteases are recognized as proteins with big potential. These proteins, termed nsP2 in Alphavirus, have the function of cleaving certain regions of the viral polyprotein, being vital to the viral cycle. In this research, we used docking and molecular dynamics to analyze the contact between the protease nsP2 of Alphavirus Chikungunya and Mayaro and substrates formed by peptides with ten amino acid residues. A model of the Mayaro nsP2 was constructed based on homologous proteases. Our study suggests that the glycine specificity motif, a region where a highly conserved glycine residue in position P2 of the protease substrate is positioned, facilitates the nucleophilic attack by assisting in placing the P1 carbonyl group carbon. Stabilization of different substrate regions maybe explained by relevant contacts with the enzyme. Besides that, the phi and psi angles in the outlier region of the Ramachandran plot found for the P2 glycine of the Chikungunya substrate seems to indicate the necessity of this residue that can accommodate angles not allowed to other residues.Communicated by Ramaswamy H. Sarma.

Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner.

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches.

Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold.

Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Mutations at the Alphavirus E1'-E2 Interdimer Interface Have Host-Specific Phenotypes.

Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane, and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at the following two interfaces: (i) the intradimer interactions between E2 and E1 of the same heterodimer and (ii) the interdimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1'). We hypothesized that the interdimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (chikungunya piggyback [CPB]) where we replaced six interdimeric residues in the E2 protein of Sindbis virus (wild-type [WT] SINV) with those from the E2 protein from chikungunya virus and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to the capsid protein and contained defects in particle morphology compared with virus derived from mosquito cells. Using cryo-electron microscopy (cryo-EM), we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the interdimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. IMPORTANCE Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as emerging pathogens, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This finding is important because it identifies a region in the spike that could be a target for antiviral drug design.

Mayaro Virus Non-Structural Protein 2 Circumvents the Induction of Interferon in Part by Depleting Host Transcription Initiation Factor IIE Subunit 2.

Mayaro virus (MAYV) is an emerging mosquito-transmitted virus that belongs to the genus Alphavirus within the family Togaviridae. Humans infected with MAYV often develop chronic and debilitating arthralgia and myalgia. The virus is primarily maintained via a sylvatic cycle, but it has the potential to adapt to urban settings, which could lead to large outbreaks. The interferon (IFN) system is a critical antiviral response that limits replication and pathogenesis of many different RNA viruses, including alphaviruses. Here, we investigated how MAYV infection affects the induction phase of the IFN response. Production of type I and III IFNs was efficiently suppressed during MAYV infection, and mapping revealed that expression of the viral non-structural protein 2 (nsP2) was sufficient for this process. Interactome analysis showed that nsP2 interacts with DNA-directed RNA polymerase II subunit A (Rpb1) and transcription initiation factor IIE subunit 2 (TFIIE2), which are host proteins required for RNA polymerase II-mediated transcription. Levels of these host proteins were reduced by nsP2 expression and during infection by MAYV and related alphaviruses, suggesting that nsP2-mediated inhibition of host cell transcription is an important aspect of how some alphaviruses block IFN induction. The findings from this study may prove useful in design of vaccines and antivirals, which are currently not available for protection against MAYV and infection by other alphaviruses.

Coordination of -1 programmed ribosomal frameshifting by transcript and nascent chain features revealed by deep mutational scanning.

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of -1PRF is coordinated by a 'slippery' sequence in the transcript, an adjacent RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop reduce the efficiency of -1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of -1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

The C-Terminal Domain of Salmonid Alphavirus Nonstructural Protein 2 (nsP2) Is Essential and Sufficient To Block RIG-I Pathway Induction and Interferon-Mediated Antiviral Response.

Salmonid alphavirus (SAV) is an atypical alphavirus that has a considerable impact on salmon and trout farms. Unlike other alphaviruses, such as the chikungunya virus, SAV is transmitted without an arthropod vector, and it does not cause cell shutoff during infection. The mechanisms by which SAV escapes the host immune system remain unknown. By studying the role of SAV proteins on the RIG-I signaling cascade, the first line of defense of the immune system during infection, we demonstrated that nonstructural protein 2 (nsP2) effectively blocks the induction of type I interferon (IFN). This inhibition, independent of the protease activity carried by nsP2, occurs downstream of IRF3, which is the transcription factor allowing the activation of the IFN promoter and its expression. The inhibitory effect of nsP2 on the RIG-I pathway depends on the localization of nsP2 in the host cell nucleus, which is linked to two nuclear localization sequences (NLS) located in its C-terminal part. The C-terminal domain of nsP2 by itself is sufficient and necessary to block IFN induction. Mutation of the NLS of nsP2 is deleterious to the virus. Finally, nsP2 does not interact with IRF3, indicating that its action is possible through a targeted interaction within discrete areas of chromatin, as suggested by its punctate distribution observed in the nucleus. These results therefore demonstrate a major role for nsP2 in the control by SAV of the host cell's innate immune response. IMPORTANCE The global consumption of fish continues to rise, and the future demand cannot be met by capture fisheries alone due to limited stocks of wild fish. Aquaculture is currently the world's fastest-growing food production sector, with an annual growth rate of 6 to 8%. Recurrent outbreaks of SAV result in significant economic losses with serious environmental consequences for wild stocks. While the clinical and pathological signs of SAV infection are fairly well known, the molecular mechanisms involved are poorly described. In the present study, we focus on the nonstructural protein nsP2 and characterize a specific domain containing nuclear localization sequences that are critical for the inhibition of the host innate immune response mediated by the RIG-I pathway.

Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein.

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

nsP4 Is a Major Determinant of Alphavirus Replicase Activity and Template Selectivity.

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis-active sequences. Here, we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123, while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s, while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV, or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. IMPORTANCE A key element of every virus with an RNA genome is the RNA replicase. Understanding the principles of RNA replicase formation and functioning is therefore crucial for understanding and responding to the emergence of new viruses. Reconstruction of the replicases of nine alphaviruses from nsP4 and P123 polyproteins revealed that the nsP4 of the majority of alphaviruses, including the mosquito-specific Eilat virus, could form a functional replicase with P123 originating from a different virus, and the corresponding chimeric viruses were replication-competent. nsP4 also had an evident role in determining the template RNA preference and the efficiency of RNA synthesis. The revealed broad picture of the compatibility of the replicase components of alphaviruses is important for understanding the formation and functioning of the alphavirus RNA replicase and highlights the possibilities for recombination between different alphavirus species.

The Putative Roles and Functions of Indel, Repetition and Duplication Events in Alphavirus Non-Structural Protein 3 Hypervariable Domain (nsP3 HVD) in Evolution, Viability and Re-Emergence.

Alphavirus non-structural proteins 1-4 (nsP1, nsP2, nsP3, and nsP4) are known to be crucial for alphavirus RNA replication and translation. To date, nsP3 has been demonstrated to mediate many virus-host protein-protein interactions in several fundamental alphavirus mechanisms, particularly during the early stages of replication. However, the molecular pathways and proteins networks underlying these mechanisms remain poorly described. This is due to the low genetic sequence homology of the nsP3 protein among the alphavirus species, especially at its 3' C-terminal domain, the hypervariable domain (HVD). Moreover, the nsP3 HVD is almost or completely intrinsically disordered and has a poor ability to form secondary structures. Evolution in the nsP3 HVD region allows the alphavirus to adapt to vertebrate and insect hosts. This review focuses on the putative roles and functions of indel, repetition, and duplication events that have occurred in the alphavirus nsP3 HVD, including characterization of the differences and their implications for specificity in the context of virus-host interactions in fundamental alphavirus mechanisms, which have thus directly facilitated the evolution, adaptation, viability, and re-emergence of these viruses.

Infection of Mammals and Mosquitoes by Alphaviruses: Involvement of Cell Death.

Alphaviruses, such as the chikungunya virus, are emerging and re-emerging viruses that pose a global public health threat. They are transmitted by blood-feeding arthropods, mainly mosquitoes, to humans and animals. Although alphaviruses cause debilitating diseases in mammalian hosts, it appears that they have no pathological effect on the mosquito vector. Alphavirus/host interactions are increasingly studied at cellular and molecular levels. While it seems clear that apoptosis plays a key role in some human pathologies, the role of cell death in determining the outcome of infections in mosquitoes remains to be fully understood. Here, we review the current knowledge on alphavirus-induced regulated cell death in hosts and vectors and the possible role they play in determining tolerance or resistance of mosquitoes.