Systematic studies are of paramount importance to identify virus–host protein–protein interactions (PPIs), since they provide an unbiased view of molecular complexes and therefore insights into viral mechanisms of replication, host resources exploitation and immune evasion strategies [11
]. Applying AP-MS in a systematic and comparative fashion can help elucidate similarities and differences in virus-specific replication strategies and even pathogenesis determinants [9
]. A few studies recently reported combined proteomic and gene perturbation approaches integrating complementary methods to investigate the cellular interactome of each of the 10 ZIKV proteins (Figure 2
a). A discrete number of previously reported bona fide interactors were consistently identified across all these studies, and appear to be shared across different flavivirus members, highlighting the relatively high degree of conservation within the genus and across diverse cellular backgrounds, thereby revealing cellular components critically required for virus replication. However, these studies also revealed specificities, reflecting the different methodologies, cellular backgrounds, enrichment strategies, gene-delivery methods and experimental designs used. For instance, using an AP-LC-MS/MS (affinity purification coupled with liquid chromatography and tandem mass spectrometry) approach in SK-N-BE2 neuroblastoma cells [34
], we identified in the ZIKV capsid interactome a strong enrichment in nuclear and nucleolar-resident proteins such as nucleolin (NCL), nucleolar RNA-dependent helicases of the DDX family, core histones (H2A), as well as peroxisomal proteins including Pex19, that have been previously reported as cellular targets of Dengue (DENV) and West Nile virus (WNV) (Figure 2
]. Interestingly, among the capsid-specific interactors, a completely new set of proteins associating with the ZIKV capsid was identified. Among these, a poorly characterized nucleolar protein involved in cell-growth regulation and maintenance of stem cell identity called LYAR was identified [38
]. LYAR was recently implicated in viral transcription and replication of Influenza A virus (IAV) through interaction with the viral ribonucleoprotein [39
], indicating a more global role of this protein in virus–host interactions. Additionally, several members of the LARP family (e.g., LARP1 and LARP7) and ZC3HAV1 (also known as ZAP) were identified as specific capsid interactors, suggesting the propensity of ZIKV capsid to interact with RNA-binding proteins. Importantly the functional relevance of some of these ZIKV capsid interactors was underlined by knock-down studies, confirming a strong reduction of viral replication upon gene depletion. In similar studies, Coyaud and collegues [40
] as well as Shah et al. [41
] confirmed these observations, identifying NCL, LYAR, LARP1, several members of the DDX family and ZC3HAV1, among the specific ZIKV capsid interactors using BioID- and AP-LC-MS/MS in 293T cells, respectively. Furthermore, recent work by Li et al. reported analogous enrichment for LARP1, LARP7 and ZC3HAV1 in a global proteomic survey for WNV-interacting proteins [42
]. Complementary functional experiments validated a conserved role for ZC3HAV1 in restricting flaviviruses since depletion of ZC3HAV1 led to a 4- to 8-fold increase in ZIKV, DENV and WNV virus titers. Interestingly, the antiviral activity of ZC3HAV1 has been investigated for several virus families including human immunodeficiency virus (HIV) and Sindbis virus (SINV) [43
]. The protein appears to recruit cellular RNA degradation machineries to specific ZAP-responsive elements on the viral mRNAs as well as the DCP1–DCP2 complex to initiate 5′-3′ RNA degradation of viral mRNA. Its activity has been mechanistically studied in the context of Japanese encephalitis virus, whereby ZC3HAV1 has been described to recruit the 3′-5′ degradation machinery [45
]. However, the exact mechanisms of virus restriction by ZC3HAV1 remain to be elucidated.
Among the other novel capsid interactors, additional work by the Ott and the Ramage groups identified the nonsense-mediated decay (NMD) pathway and the exon-junction complex (EJC) as targets of the WNV, DENV and ZIKA virus capsids [42
]. While the antiviral activity of individual members of these complexes appears moderate in knock-down studies, further experimental evidence supports a direct interaction between the viral RNA and RBM8A (a central component of the EJC), suggesting a depletion of the cellular PYM1 pool upon recruitment by the WNV capsid to protect viral RNA from decay. Similarly, direct involvement of the NMD pathway in flavivirus is corroborated by the identification of the regulator of nonsense transcripts 1 (UPF1) among the ZIKV capsid interactors and the upregulation of several NMD substrate mRNAs upon ZIKV infection both in Huh7 and hNPC. Furthermore, depletion of UPF1 leads to a 50% increase in virus replication, supporting the notion that NMD acts as flavivirus restriction machinery that is counteracted by viral activities [46
In addition to proteins recruited by several flavivirus capsids, the ZIKV capsid is the only one reported to specifically associate with cellular factors that are involved in neuronal development or neurological disorders [34
]. These proteins include neuroguidin (NGDN), an eIF4E-interacting protein mediating CPEB-mediated translation of essential genes in early development and neuronal synaptic plasticity. Further ZIKA capsid interactors are several members of the survival motor neuron (SMN) complex [34
], which is important for proper splicing, neuronal migration and differentiation. It would be interesting to assess binding specificity of other flavivirus capsid proteins in a similar neuronal background and investigate further their contribution to viral replication or pathogenesis. Such studies could pinpoint novel viral pathogenesis determinants and assist in the identification of druggable binding surfaces required for specific protein–protein interactions.
Altogether, flavivirus capsids evidently evolved evasive mechanisms through binding/sequestration of ZC3HAV1 and other RNA-binding proteins (e.g., UPF1, LARP7, NGDN). Capsid proteins are apparently exploiting their versatile subcellular localization (both nuclear and cytoplasmic) and their intrinsic hydrophobic character to simultaneously highjack and diverge regulation or expression of specific transcripts.