The alphaviruses (family Togaviridae
) are widely spread all over the globe, found on every continent, and have been identified not only in different vertebrate hosts such as mammals, birds and fish, but also in insects. There are several human and animal pathogens in the alphavirus genus [1
]. The majority of alphaviruses are transmitted between different vertebrate species by various mosquitoes [1
]. Mosquito-transmitted alphaviruses can be divided into Old and New World alphaviruses. Chikungunya virus (CHIKV), belonging to the Old World alphaviruses, has relatively recently emerged in Asia, Africa and America and its infection results in severe illness in humans [5
]. Semliki Forest virus (SFV) and Sindbis virus (SINV) also belong to Old World alphaviruses and have been extensively used as models for studies of alphaviruses. Most studied of the New World alphaviruses are Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) whose infections have economic and medical consequences in South and Central America. Despite the similarity of genome organization and basic replication strategy of Old and New World alphaviruses, these groups greatly differ in their host interactions, e.g., use different strategies to counteract host anti-viral responses and interact with a different set of host proteins [6
]. Significant differences in virus-host interactions have also been observed for alphaviruses belonging to the same group, for example SFV and CHIKV [8
] or VEEV and EEEV [7
The alphavirus genome is a single-stranded RNA molecule with positive polarity, its 5′ end is capped and the 3′ end has a poly(A) tail. In infected cells, genomic RNA is used as an mRNA for the translation of the large non-structural (ns) polyprotein P1234, the precursor of four mature replicase proteins nsP1, nsP2, nsP3 and nsP4 [3
]. Alphavirus RNA replication depends on P1234 expression and its highly-regulated processing. The early viral replication complex, which consists of the P123 processing intermediate and mature nsP4, synthesizes negative strand RNA using the genomic strand as template, resulting in the formation of double-stranded RNA (dsRNA) replication form [12
]. This process coincides with the formation of plasma membrane invaginations termed spherules that contain viral dsRNA, nsPs and cellular components [14
]. The processing of the P123 intermediate into mature nsP1, nsP2 and nsP3 brings an end to the synthesis of negative-strand RNA and triggers formation of late replication complexes which use viral dsRNA template to make new genomic and subgenomic RNA strands [12
]. The latter is synthesized from an internal promoter located on the negative strand RNA and its sequence corresponds to that of the last one third of the genomic RNA. Subgenomic RNA is needed for the synthesis of alphavirus structural proteins C, E3, E2, 6K, TF and E1 [17
In infected vertebrate cells, alphavirus spherules are first observed at the plasma membrane; later in infection they are often re-localised to internal membranes, such as endosomal or lysosomal membranes [11
]. nsP1 anchors the replication complexes to the membranes [22
], and is involved in the synthesis of cap-structure and RNA negative strands [24
]. nsP2 has protease activity, which is required to process P1234 into mature non-structural proteins [25
]. In addition, nsP2 is also an RNA triphosphatase involved in viral cap synthesis and has NTPase and RNA helicase activities [27
]. nsP4 is the viral RNA-dependent RNA polymerase and terminal adenosine transferase and is directly responsible for synthesis of different viral RNAs and adding poly(A) tails to positive strand RNAs [30
For a long time, the roles of nsP3 in virus infection were poorly understood. However, recent data have revealed nsP3 involvement in different alphavirus-host interactions. nsP3 can be divided into three domains: N-terminal macro domain, middle conserved domain termed alphavirus unique domain (AUD) or zinc-binding domain, and C-terminal hypervariable domain (HVD) [7
]. 3D structures have been revealed for the first two of these domains while the HVD has been predicted to be intrinsically unstructured [33
]. The macro domain can bind RNA, mono(ADP-ribose) and poly(ADP-ribose). For some alphaviruses the macro domain possesses adenosine diphosphoribose 1”-phosphate phosphatase activity, which likely gives the ability to hydrolyze ADP-ribose groups from aspartate and glutamate residues of mono(ADP-ribosyl)ated proteins [33
]. Similarly, the macro domain of distantly related hepatitis E virus can remove mono(ADP-ribose) but also poly(ADP-ribose) from proteins [37
]. This function is crucial for alphavirus infection both in vertebrate and invertebrate cells [38
]. In addition, the macro domain also coordinates P1234 processing [33
]. Many mutations in AUD are lethal for alphaviruses indicating that this region is also essential for virus infection. The functional roles of the AUD are, however, unknown.
As the name implies, the HVD is poorly conserved: with the exception of scattered short linear motifs, there is little similarity between HVDs of different alphaviruses [7
]. For several alphaviruses, such as SFV and SINV, it is known that HVD is phosphorylated [40
], the same is presumed for other alphaviruses as well. HVD is an intrinsically unstructured region, tolerates large insertions and deletions and has been shown to bind various host proteins [39
]. Indeed, binding motifs for several of such host proteins have already been identified within the domain. HVD of Old World alphaviruses bind Ras GTPase-activating protein-binding protein 1 (G3BP1) and Ras GTPase-activating protein-binding protein 2 (G3BP2) using a duplicated FGDF motif located almost at the very end of the domain, just before the degradation signal [39
]. Unlike many other interactions with host proteins, the nsP3:G3BP interaction is crucial: in the absence of these proteins (or if the interaction motifs are removed) the Old World alphaviruses replicate poorly or not at all [7
]. In addition, the nsP3:G3BP interaction disrupts stress granule formation that may also be beneficial for virus infection [46
]. nsP3 of New World alphaviruses bind different stress granule components Fragile X mental retardation 1 (FMR1), FMR1 autosomal homolog 1 (FXR1), and FMR1 autosomal homolog 2 (FXR2); this interaction is also required for efficient virus replication [7
]. Curiously, EEEV occupies the “middle ground” and can utilize both G3BP and FXR proteins to support its replication [10
]. Milder effects on alphavirus infection are observed if interactions with other host proteins are prohibited. Examples of such proteins include amphiphysins that bind via their SRC Homology 3 (SH3) domains to the polyproline-rich nsP3 region of the Old World alphaviruses [50
]. In addition, deletion of phosphorylated regions of SFV nsP3 HVD disrupted the hyper-activation of the pro-survival PI3K-Akt-mTOR pathway. Further analysis indicated the presence of a YXXM motif that is needed for binding of PI3K regulatory subunit p85, which results in Akt activation [8
]. The many of functions of the enigmatic alphavirus nsP3 proteins have been recently reviewed by Gotte et al., 2018 [52
To better understand the role of HVD in binding host cell proteins and its importance for alphavirus infection, a proteomics study was performed to systematically identify binding partners of the CHIKV nsP3 HVD. Among other proteins, CD2-associated protein (CD2AP) and SH3 domain-containing kinase-binding protein (SH3KBP1) were identified as prominent interaction partners. Both proteins co-localised with CHIKV and SFV replication complexes in infected cells. The binding site was mapped to the HVD and point mutations in this site were shown to disrupt interaction with CD2AP and SH3KBP1 as well as several functionally connected cellular proteins. The motif was found to be common, yet not universal, for both Old and New World alphaviruses. The mutant viruses lacking the motif for CD2AP and SH3KBP1 binding were infectious, albeit attenuated. Our findings indicate that the nsP3 interactions with CD2AP and SH3KBP1 (and/or interaction with proteins interacting with the same binding motif) is required for efficient infectivity of alphaviruses, but has no major impact on localisation of viral replication complexes.
2. Materials and Methods
2.1. Cell Lines
BHK-21 (baby hamster kidney, ATCC CCL-10) cells were grown in Glasgow minimum essential medium (GMEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 2% tryptose phosphate broth (TPB), 20 mM HEPES and penicillin-streptomycin (100 U/mL and 0.1 mg, respectively). HOS (human osteosarcoma, ATCC CRL-1543) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% FBS, 2mM l-glutamine, and penicillin-streptomycin. For cultivation of Flp-In T-REx 293 cells (Thermo Fisher Scientific) DMEM also contained zeocin (at 100 μg/mL) and blasticidin (at 15 μg/mL). For propagation of transgenic cell lines based on Flp-In T-REx 293 zeocin was omitted and hygromycin B (at 100 μg/mL) was used instead. All the cells were grown in a humidified incubator at 37 °C in a 5% CO2 atmosphere.
For proteomics study cell lines were labelled with stable isotopes as follows: cells were grown in SILAC DMEM (Thermo Fisher Scientific) supplemented with 10% dialyzed FBS (Thermo Fisher Scientific), heavy arginine (0.133 mM, CNLM-539; Cambridge Isotope Laboratories Inc., Tewskbury, MA, USA) and heavy lysine (0.266 mM, CNLM-291; Cambridge Isotope Laboratories Inc.), hygromycin B (at 100 μg/mL), blasticidin (at 15 μg/mL) and penicillin-streptomycin for at least five generations.
2.2. Construction of Expression Constructs for HVD Domains of Different Alphaviruses and Making of the Stable Transgenic Cell Lines
Sequences encoding Flag-tagged EGFP and Flag-tagged EGFP fused to nsP3 HVD from CHIKV of East/Central/South African genotype isolate LR2006-OPY1 (amino acid residues 327–530 of nsP3) or VEEV (residues 327–557 of nsP3) were cloned into modified pEF5/FRT/V5-DEST vector (Thermo Fisher Scientific) under the human EF-1α promoter. Sequences encoding for HVD regions of SINV (Toto1101, residues 327–556 of nsP3), O’nyong’nyong virus (ONNV) (residues 327–549 of nsP3), Barmah Forest virus (BFV) (residues 326–470 of nsP3) and Ross River virus (RRV) (T48 strain, residues 328–535 of nsP3) were cloned using the same approach.
Point mutations preventing interaction with CD2AP (P423A, R428A for CHIKV, and P428A, R433A, P435A, P439A, R440A, R444A for SFV) as well as various deletions were introduced into HVD region of nsP3 using PCR-based site-directed mutagenesis and subcloning procedures. The obtained fragments harbouring desired mutations were transferred to infectious cDNA (icDNA) clones of CHIKV or SFV4 as well as into expression constructs used for stable or transient expression of EGFP tagged with nsP3 HVD of CHIKV or SFV. All constructs were verified using Sanger sequencing, their sequences are available upon request.
To make stable transgenic cells lines 107 Flp-In T-REx 293 cells were co-transfected with 9 µg of pOG44 plasmid (Thermo Fisher Scientific) used for expression of Flp recombinase and 1 µg of expression construct for Flag-tagged EGFP, Flag-tagged EGFP fused to nsP3 HVD of VEEV, wild-type (wt) CHIKV, mutant CHIKV (P423A and R428A substitutions) or mutant SFV (P428A, R433A, P435A, P439A, R440A and R444A substitutions) using Lipofectamine 2000 reagent (Thermo Fisher Scientific). At 48 h post-transfection (p.t.) the medium was replaced with a selection medium containing hygromycin B (at 100 μg/mL); subsequently medium was changed every 3–4 days to remove dead cells. Formed hygromycin B resistant colonies were picked up and verified for the expression of intended proteins using fluorescent microscopy (for EGFP auto-fluorescence) and western blotting.
2.3. Virological Methods
Plasmids containing wt or mutant icDNA of CHIKV were linearized using NotI restriction enzyme. In vitro transcription was carried out using linearized plasmids as templates and the mMESSAGE mMACHINE SP6 transcription kit (Thermo Fisher Scientific); obtained RNA was controlled for its quality and quantified using agarose gel electrophoresis and staining with ethidium bromide.
Virus recovery was performed as follows, 8 × 106
BHK-21 cells resuspended in ice-cold PBS were transfected with 10 μg of in vitro transcribed RNAs by electroporation using a Gene Pulser II unit (two pulses at 850 V and 25 μF, Bio-Rad, Hercules, CA, USA), 48 h later virus stock was harvested to be used in further experiments. Infectious centre assay (ICA) was conducted to measure RNA infectivity, 1 μg of in vitro transcribed RNA was used and ten-fold dilutions of electroporated cells were seeded on top of 1.5 × 106
BHK-21 cells grown in the wells of a 6-well tissue culture plate. After 2 h of incubation at 37 °C, the cell culture medium was removed and the cells were overlaid with 2 mL of GMEM containing 0.8% carboxymethyl cellulose (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2% FBS. Cells were stained with crystal violet after 3 days of incubation at 37 °C. To recover SFV4 and its mutants pCMV-SFV4 plasmid was used as previously described [53
]. Electroporated cells were plated on 6 cm diameter plates and covered by growth medium. P0
virus stocks we collected upon development of cytopathic effects.
The titres of obtained virus stocks and growth curve samples were determined using standard plaque assays, titre given in plaque forming units per mL (PFU/mL). For this ten-fold serial dilutions of collected stocks were prepared and used to infect BHK-21 growing in wells of 6-well plates at 90–100% confluency. 1 h post-infection (p.i.) infectious media were aspirated and cells were overlaid with 2 mL of GMEM containing 2% FBS and 0.8% carboxymethyl cellulose. Cells were fixed 3 days p.i., stained with crystal violet and plaques were counted.
2.4. Immunoprecipitation and Proteomics Analysis
3 × 107
cells were harvested for proteomics studies; 106
cells were used for co-immunoprecipitation experiments with alphavirus HVD-tagged EGFP. Cells were centrifuged and washed once with cold PBS. Harvested cells were lysed with ice-cold lysis buffer (150 mM NaCl, 20 mM Hepes (at pH 7.2), 100 mM K-acetate, 2 mM MgCl2
, 0.1% Tween-20, 1% Triton X-100, 1 Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland) per 50 mL) on ice for 30 min, non-lysed material was removed by centrifugation at 15,000× g
for 20 min at 4 °C; the obtained supernatant was transferred into a new tube. Magnetic beads for binding EGFP (GFP-trap M, Chromotek, Planegg, Germany) were equilibrated with lysis buffer and added to the supernatant and incubated at 4 °C for 1 h keeping the tubes constantly rotating. The beads were then washed four times with the lysis buffer and material bound to beads was eluted by boiling in 1% SDS solution (for proteomics samples) or in 1× Laemmli buffer (for immunoblot analysis). For proteomics samples, proteins were precipitated with 10% TCA and resolubilized in 7 M urea/2 M thiourea, 100 mM ammonium bicarbonate (ABC) buffer. After disulphide reduction with 5 mM dithiothreitol and cysteine alkylation with 10 mM iodoacetamide, proteins were in-solution digested for 4 h at room temperature with 1:50 (enzyme to protein) Lys-C (Wako, Richmond, VA, USA, diluted 5-times with 100 mM ABC, and further digested overnight with 1:50 dimethylated trypsin (Sigma-Aldrich). Samples were desalted on an in-house made C-18 tips. Peptides were separated on an Ultimate 3000 RSLCnano system (Dionex, Sunnyvale, CA, USA) using a C18 cartridge trap-column in a backflush configuration and an in-house packed (3 µm C18 particles, Dr Maisch, Ammerbuch, Germany) analytical 50 cm × 75 µm emitter-column (New Objective, Woburn, MA, USA). Peptides were eluted at 200 nL/min with an 8–40% of B 90 min gradient (buffer B: 80% acetonitrile + 0.1% formic acid, buffer A: 0.1% formic acid) to a Q Exactive Plus (Thermo Fisher Scientific) tandem mass spectrometer operating with a top-5 strategy and with a maximum cycle time of 0.9 s. Briefly, one 350–1400 m/z MS scan at a resolution of R = 70,000 was followed by higher-energy collisional dissociation fragmentation (normalized collision energy of 26) of the 5 most-intense ions (z: +2 to +6) at R = 17,500. The MS and MS/MS ion target values and injection times were 3 × 106
, 50 ms and 5 × 104
, 110 ms respectively. Dynamic exclusion was limited to 25 s. Obtained raw LC/MS/MS data was identified and quantified using the MaxQuant software package [54
]. Arg10 and Lys8 were defined as the heavy amino acids and spectra were searched against UniProt (http://www.uniprot.org
) Homo sapiens
reference proteome supplemented with CHIKV and SFV nsP3 sequences. Minimally two peptides at least seven amino acids long were required for identification of a protein. All other parameters were default. Two-fold difference from mean was taken as a cut-off value. Functional analysis of host proteins was conducted using the STRING database (string-db.org).
Immunoprecipitation of CHIKV or SFV nsP3 protein from infected cells was performed as described before [46
]. Briefly, 2.4 × 106
cells were washed with ice-cold PBS and lysed with ice-cold lysis buffer (20 mM HEPES, pH 7.4, 110 mM potassium acetate, 2 mM MgCl2
, 0.1% Tween 20, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5 M NaCl) supplemented with protease inhibitor on ice for 10 min. Cell debris was removed from the lysates by centrifugation at 14,000× g
for 10 min at 4 °C; the obtained supernatant was transferred to a new tube and divided into two fractions, 30 µL for whole-cell lysates and 550 µL for co-immunoprecipitation assays. For co-immunoprecipitation experiments, supernatant was incubated with primary antibody (rabbit anti-SFV or anti-CHIKV nsP3, both in house) for 15 min at room temperature in constant rotation. Magnetic beads (Protein G, GE Healthcare, Chicago, IL, USA) were equilibrated with lysis buffer, added to the supernatant and incubated overnight at 4 °C in constant rotation. The beads were then washed four times with lysis buffer and proteins were eluted by heating in 1× NuPAGE LDS sample buffer (Thermo Fisher Scientific) supplemented with DTT for 10 min at 80 °C.
2.5. Immunoblot Analysis
Protein samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against EGFP (rabbit, in house), nsP3 of SFV or CHIKV (both rabbit, in house), β-actin (mouse, sc-47778; or goat, sc-1616; Santa Cruz Biotechnology, Dallas, TX, USA) or CD2AP (mouse, (B4) sc-25272, or rabbit, (H290) sc-9137, both Santa Cruz Biotechnology). Species-specific horseradish peroxidase (HRP) conjugated secondary antibodies (LabAs Ltd., Tartu, Estonia) and enhanced chemiluminescence reagents were used to develop the blots as described previously [9
2.6. Immunofluorescence Microscopy
Cells were grown on coverslips in 35 mm dishes. At 50% confluence, the cells were infected with wt CHIKV, SFV or their mutant versions harbouring substitutions mutations in nsP3 at an MOI 10, mock-infected cells were used as a control. At selected time points, the cells were fixed with 4% paraformaldehyde for 10 min and subsequently permeabilized with 0.5% Triton X-100 for 3 min. Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary). Incubation with primary antibody was followed by incubation with secondary anti-mouse or anti-rabbit antibodies conjugated to Alexa Fluor555 or Alexa Fluor488 (Thermo Fisher Scientific). Draq5 (BioStatus, Loughborough, UK) was used to counterstain nuclei. Washed coverslips were mounted in mounting medium and images were obtained and analysed using a LSM710 confocal microscope (Zeiss, Oberkochen, Germany) or TCS SP5 X microscope (Leica, Wetzlar, Germany) equipped with a super continuum pulsed white laser. Images were processed using Photoshop (Adobe Systems Incorporated, San Jose, CA, USA).
Recent studies have changed the status of alphavirus nsP3 from an enigmatic component to that of a protein with many important roles [52
]. For example, it is involved in removing mono(ADP-ribosyl)ation from host proteins counteracting an immune response and regulating viral RNA synthesis [38
]. In addition, the nsP3 hypervariable and presumably unstructured C-terminal region has turned out to be a hub for various host-virus interactions. Some of these are not crucial for virus replication and therefore nsP3 can tolerate significant modifications in its HVD up to almost complete deletion or replacement with heterologous protein sequence [7
]; it also tolerates insertion of protein-tags in several positions in the HVD. On the other hand, some interactions, such as those with stress granule proteins are crucial, possibly due to their primary role in allowing the assembly of functional replication complexes [7
]. Since the HVD is intrinsically unstructured, these interactions are likely mediated by relatively short linear sequence motifs located in HVD. Indeed, multiple sequence alignment allows to detect common motifs shared between HVD of different alphaviruses even though the lengths and especially the sequences of this region vary greatly [7
In this study, we expressed nsP3 HVD domains of different alphaviruses as fusion proteins with EGFP, generated several stable human cell lines and used these to capture and identify cellular protein interactors of CHIKV, VEEV and SFV HVD. The dataset we obtained for CHIKV and VEEV nsP3 HVD showed good overlap with datasets published previously, even though those were obtained using different approaches. In these previous studies, BHK-21 or Vero cells were used, either infected with SINV expressing EGFP-tagged nsP3 [43
] or alternatively, infected with alphavirus or replicons expressing EGFP-fused HVD from a subgenomic promoter [7
]. The good overlap serves as validation of the approach used in this study and our dataset also confirms the previous findings. We were also able to identify several proteins interacting with CHIKV nsP3 HVD, for example, PARP1, previously shown to bind nsP3 HVD of SINV [56
], also G3BP1 and G3BP2 [7
The roles of many of the nsP3 interacting proteins, identified in this and other studies, for alphavirus infection remain poorly understood. Among the proteins with an undescribed role in virus infection, CD2AP was one of the most prominent, at least when judged by its relative abundance in our CHIKV and VEEV nsP3 HVD interactors dataset. Further analysis using immunoblotting readily confirmed its interaction with CHIKV nsP3 HVD and allowed mapping of the interaction site; we confirmed that the binding occurs via a previously uncharacterized SLIM, termed M2. Immunofluorescence studies showed that in CHIKV-infected cells, CD2AP co-localised with nsP3 and dsRNA, and that its interaction with virus replication complex components was solely dependent on the motif M2 located in nsP3. Motifs similar to M2 were also found to be present in the nsP3 HVD of many other alphaviruses, and more importantly, experiments confirmed their nsP3 HVD interaction with CD2AP. The motif was also essential for binding to SH3KBP1, a related protein to CD2AP. Interestingly, the prototype member of genus alphavirus, SINV, lacks this motif as well as the ability of its nsP3 to interact with CD2AP, confirming findings recently reported by others [10
]. The implications of this unique situation with SINV are not known; either this virus uses some other interactor(s) to substitute CD2AP or its replication does not require or benefit from this kind of interaction partner. Indeed, VEEV nsP3 does not bind G3BP1 and G3BP2 but FMR1, FXR1, FXR2 proteins instead and these interactions have been shown to be essential for virus replication [7
]. Our study did reveal that nsP3 interaction with CD2AP and SH3KBP1 is indeed important for alphaviruses, and although both SFV and CHIKV tolerate mutations abolishing these interactions relatively well, it results in reduction of CHIKV multiplication. Similarly, motif M1 in the current that has SH3 ligand (398-PVAPPRRR-406) is known to bind Amphiphysin-1 and Amphiphysin-2, which have membrane-bending properties, abolishing this interaction results in attenuated SFV [50
]. Our methodology was more suitable to capture cytoplasmic proteins, as evidenced by our failure to capture amphiphysins. The close vicinity of two SH3 ligands (in M1 and M2) may indicate that they are also functionally linked, a matter for further studies.
CD2AP is one of the organizers of cellular actin cytoskeleton [57
]. One of the roles of CD2AP is to cap actin fibres together with CAPZA1, CAPZA2, CAPZB proteins. CD2AP and SH3KBP1, which is homologous to CD2AP, play versatile role, involved in dynamic actin cytoskeleton remodelling. These functions include recruitment of above listed actin capping proteins, it can facilitate branched actin filament formation [61
] and has actin bundling properties [57
]. Also CD2AP and SH3KBP1 have been shown to be important for receptor-mediated endocytosis and endosome formation [57
]. However, when the motif was mutated in SFV or CHIKV we could not detect markedly altered replication complex localisation in the cells, however, there is a possibility that the lack of such interactions leads to altered replication complex stability. It is important to note that CD2AP and SH3KBP1 are likely not the only cellular proteins for which binding is mediated by motif M2. Other cytoskeleton components were also pulled down by CHIKV and VEEV nsP3 HVD, among them were also actin capping proteins CAPZA1, CAPZA2, CAPZB. Their interaction with the HVD was lost when mutations preventing interaction with CD2AP were introduced into SFV nsP3, indicating that these proteins interact with the same motif and/or with each other. Therefore, it would be important to elucidate their individual or combined roles during alphavirus infection. However, multiple protein partners prevent simple and straightforward interpretation of the data, e.g., assigning all discovered effects solely to the CD2AP binding. It is more likely that CD2AP and SH3KBP1 directly bind to nsP3 motif M2 via their SH3 domain; this domain is missing in CAPZA1, CAPZA2 and CAPZB, their binding may be mediated by other proteins including CD2AP and/or SH3KBP1. The possible functional redundancy also severely limits use of CRISPR-Cas knock-out technology as simultaneous knock-out (or even knock-down) of such a large number of host genes is challenging.
Also, it is interesting that VEEV nsP3 HVD binds the same set of cytoskeleton proteins as CHIKV and SFV. However, it is not clear if these interactions have the same role in VEEV infection as for CHIKV. It is interesting to mention that while our analysis revealed CD2AP, SH3KBP1, CAPZB, CAPZA1 and CAPZA2 as top interaction partners both for CHIKV and VEEV, a recent study found them to interact more prominently with nsP3 of VEEV; in fact CAPZA1 and CAPZA2 were not detected as interaction partners for nsP3 of CHIKV in that study [10
]. The reasons for this discrepancy can be attributed to the use of a different cell line and/or different protein capture approaches. However, it may also indicate that interaction with this set of proteins is more prominent (or more stable) and therefore possibly more important for VEEV. Thus, it is an important topic for further studies to control if the replication of New World alphaviruses is dependent on their ability to bind CD2AP and the other cytoskeleton components.
It should also be emphasized that while this study focused mostly on one motif in nsP3 HVD, there are potentially other still uncharacterized motifs in HVD that are involved in interactions with host proteins, for example the seven last amino acids in the M1 (407-GRNLTVT-413) of CHIKV. This generates a possibility for the presence of a complicated network, where the effect of one virus-host interaction is likely modified by the presence or absence of several other interactions. Unravelling this network, revealing functional significance of such interactions and their combinations not only for virus replications in cell culture but also in vivo, represent significant challenges for additional studies. Due to the flexibility of nsP3 HVD it can act as an evolutionary “playground”—new motifs to bind new partners can easily evolve, increasing the chances that the virus can evade restricting factors. Additionally, the results of studies of SLIMs can give valuable information to design better vaccine candidates [63
] as mutating those motifs can help to produce weakened viruses.