What is the functional reach of wastewater surveillance for respiratory viruses, pathogenic viruses of concern, and bacterial antibiotic resistance genes of interest?

BACKGROUND: Despite a clear appreciation of the impact of human pathogens on community health, efforts to understand pathogen dynamics within populations often follow a narrow-targeted approach and rely on the deployment of specific molecular probes for quantitative detection or rely on clinical detection and reporting. MAIN TEXT: Genomic analysis of wastewater samples for the broad detection of viruses, bacteria, fungi, and antibiotic resistance genes of interest/concern is inherently difficult, and while deep sequencing of wastewater provides a wealth of information, a robust and cooperative foundation is needed to support healthier communities. In addition to furthering the capacity of high-throughput sequencing wastewater-based epidemiology to detect human pathogens in an unbiased and agnostic manner, it is critical that collaborative networks among public health agencies, researchers, and community stakeholders be fostered to prepare communities for future public health emergencies or for the next pandemic. A more inclusive public health infrastructure must be built for better data reporting where there is a global human health risk burden. CONCLUSIONS: As wastewater platforms continue to be developed and refined, high-throughput sequencing of human pathogens in wastewater samples will emerge as a gold standard for understanding community health.

Microbiome-mediated incapacitation of interferon lambda production in the oral mucosa.

Here, we show that Porphyromonas gingivalis (Pg), an endogenous oral pathogen, dampens all aspects of interferon (IFN) signaling in a manner that is strikingly similar to IFN suppression employed by multiple viral pathogens. Pg suppressed IFN production by down-regulating several IFN regulatory factors (IRFs 1, 3, 7, and 9), proteolytically degrading STAT1 and suppressing the nuclear translocation of the ISGF3 complex, resulting in profound and systemic repression of multiple interferon-stimulated genes. Pg-induced IFN paralysis was not limited to murine models but was also observed in the oral tissues of human periodontal disease patients, where overabundance of Pg correlated with suppressed IFN generation. Mechanistically, multiple virulence factors and secreted proteases produced by Pg transcriptionally suppressed IFN promoters and also cleaved IFN receptors, making cells refractory to exogenous IFN and inducing a state of broad IFN paralysis. Thus, our data show a bacterial pathogen with equivalence to viruses in the down-regulation of host IFN signaling.

Protein Kinase C subtype δ interacts with Venezuelan equine encephalitis virus capsid protein and regulates viral RNA binding through modulation of capsid phosphorylation.

Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis.

Topical Oral and Intranasal Antiviral Agents for Coronavirus Disease 2019 (COVID-19).

With the largest viral loads in both symptomatic and asymptomatic patients with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) present in the oral and nasal cavities, agents that act on these two areas have the potential for large therapeutic and prophylactic benefit. A literature review was conducted to elucidate the possible agents useful in treatment of SARS-CoV-2. These agents were evaluated for their current applications, adverse reactions, their current state of study, and any future considerations in their management of coronavirus disease 2019 (COVID-2019). Our review has found that, while there are many promising agents with proven efficacy in their in-vitro efficacy against SARS-CoV-2, more clinical trials and in-vivo studies, as well as safety trials, must be conducted before these agents can be effectively implemented.

Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions.

Arthropod-borne viruses, such as the members of the genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish and complete their life cycles. Despite considerable efforts to define the host-pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA-protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduce a robust viral RNA-protein discovery method to elucidate the Sindbis virus (SINV) RNA-protein host interface. Cross-link-assisted mRNP purification (CLAMP) assessment revealed an extensive array of host-pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of protein-vRNA interaction by a UV cross-linking and immunoprecipitation sequencing (CLIP-seq) approach and assessed the consequences of the RNA-protein binding event of hnRNP K, hnRNP I, and hnRNP M in regard to viral infection. Here, we demonstrate that mutation of the prioritized hnRNP-vRNA interaction sites effectively disrupts hnRNP-vRNA interaction. Correlating with disrupted hnRNP-vRNA binding, SINV growth kinetics were reduced relative to wild-type parental viral infections in vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics was found to be dysregulated structural-gene expression. Collectively, this study further defines the scope and importance of the alphavirus host-pathogen vRNA-protein interactions.IMPORTANCE Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their life cycle. However, the extent and importance of host-pathogen RNA-protein interactions are woefully undercharacterized. The efforts detailed in this study fill this critical gap, and the significance of this research is 3-fold. First, the data presented here fundamentally expand the scope and understanding of alphavirus host-pathogen interactions. Second, this study identifies the sites of interaction for several prioritized interactions and defines the contribution of the RNA-protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of the given host-pathogen interactions may be assessed in the future, using a mouse model of infection.

Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants.

Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant.

Alphavirus RNA synthesis and non-structural protein functions.

The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.

Encapsidation of host-derived factors correlates with enhanced infectivity of Sindbis virus.

The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV(Heavy), is more infectious on a per-particle basis than SINV(Light). SINV produced in mosquito cell lines (SINV(C6/36)) exhibited particle-to-PFU ratios similar to those observed for SINV(Heavy). In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV(Heavy) or SINV(C6/36) than following infection with SINV(Light), due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV(Heavy) and SINV(C6/36) contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles.

Capsid protein mediated evasion of IRAK1-dependent signalling is essential to Sindbis virus neuroinvasion and virulence in mice.

ABSTRACTAlphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses that are recognized as rapidly emerging pathogens. Despite being exquisitely sensitive to the effects of the innate immune response alphaviruses can readily replicate, disseminate, and induce pathogenesis in immunologically competent hosts. Nonetheless, how alphaviruses evade the induction of an innate immune response prior to viral gene expression, or in non-permissive infections, is unknown. Previously we reported the identification of a novel host/pathogen interaction between the viral Capsid (CP) protein and the host IRAK1 protein. The CP/IRAK1 interaction was determined to negatively impact IRAK1-dependent PAMP detection in vitro, however, the precise importance of the CP/IRAK1 interaction to alphaviral infection remained unknown. Here we detail the identification of the CP/IRAK1 interaction determinants of the Sindbis virus (SINV) CP protein and examine the importance of the interaction to alphaviral infection and pathogenesis in vivo using an interaction deficient mutant of the model neurotropic strain of SINV. Importantly, these interaction determinants are highly conserved across multiple Old-World alphaviruses, including Ross River virus (RRV), Mayaro virus (MAYV), Chikungunya virus (CHIKV), and Semliki Forest virus (SFV). In the absence of a functional CP/IRAK1 interaction, SINV replication is significantly restricted and fails to disseminate from the primary site of inoculation due to the induction of a robust type-I Interferon response. Altogether these data indicate that the evasion of IRAK1-dependent signalling is critical to overcoming the host innate immune response and the in vivo data presented here demonstrate the importance of the CP/IRAK1 interaction to neurovirulence and pathogenesis.

Extending the coverage of spectral libraries: a neighbor-based approach to predicting intensities of peptide fragmentation spectra.

Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well-studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor-based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K-nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20-60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.

Dephosphorylation of HuR protein during alphavirus infection is associated with HuR relocalization to the cytoplasm.

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

Mutating conserved cysteines in the alphavirus e2 glycoprotein causes virus-specific assembly defects.

There are 80 trimeric, glycoprotein spikes that cover the surface of an alphavirus particle. The spikes, which are composed of three E2 and E1 glycoprotein heterodimers, are responsible for receptor binding and mediating fusion between the viral and host-cell membranes during entry. In addition, the cytoplasmic domain of E2 interacts with the nucleocapsid core during the last stages of particle assembly, possibly to aid in particle stability. During assembly, the spikes are nonfusogenic until the E3 glycoprotein is cleaved from E2 in the trans-Golgi network. Thus, a mutation in E2 potentially has effects on virus entry, spike assembly, or spike maturation. E2 is a highly conserved, cysteine-rich transmembrane glycoprotein. We made single cysteine-to-serine mutations within two distinct regions of the E2 ectodomain in both Sindbis virus and Ross River virus. Each of the E2 Cys mutants produced fewer infectious particles than wild-type virus. Further characterization of the mutant viruses revealed differences in particle morphology, fusion activity, and polyprotein cleavage between Sindbis and Ross River virus mutants, despite the mutations being made at corresponding positions in E2. The nonconserved assembly defects suggest that E2 folding and function is species dependent, possibly due to interactions with a virus-specific chaperone.

Dancing with the Devil: A Review of the Importance of Host RNA-Binding Proteins to Alphaviral RNAs during Infection.

Alphaviruses are arthropod-borne, single-stranded positive sense RNA viruses that rely on the engagement of host RNA-binding proteins to efficiently complete the viral lifecycle. Because of this reliance on host proteins, the identification of host/pathogen interactions and the subsequent characterization of their importance to viral infection has been an intensive area of study for several decades. Many of these host protein interaction studies have evaluated the Protein:Protein interactions of viral proteins during infection and a significant number of host proteins identified by these discovery efforts have been RNA Binding Proteins (RBPs). Considering this recognition, the field has shifted towards discovery efforts involving the direct identification of host factors that engage viral RNAs during infection using innovative discovery approaches. Collectively, these efforts have led to significant advancements in the understanding of alphaviral molecular biology; however, the precise extent and means by which many RBPs influence viral infection is unclear as their specific contributions to infection, as per any RNA:Protein interaction, have often been overlooked. The purpose of this review is to summarize the discovery of host/pathogen interactions during alphaviral infection with a specific emphasis on RBPs, to use new ontological analyses to reveal potential functional commonalities across alphaviral RBP interactants, and to identify host RBPs that have, and have yet to be, evaluated in their native context as RNA:Protein interactors.

Male microbiota-associated metabolite restores macrophage efferocytosis in female lupus-prone mice via activation of PPARγ/LXR signaling pathways.

Systemic lupus erythematosus development is influenced by both sex and the gut microbiota. Metabolite production is a major mechanism by which the gut microbiota influences the immune system, and we have previously found differences in the fecal metabolomic profiles of lupus-prone female and lupus-resistant male BWF1 mice. Here we determine how sex and microbiota metabolite production may interact to affect lupus. Transcriptomic analysis of female and male splenocytes showed genes that promote phagocytosis were upregulated in BWF1 male mice. Because patients with systemic lupus erythematosus exhibit defects in macrophage-mediated phagocytosis of apoptotic cells (efferocytosis), we compared splenic macrophage efferocytosis in vitro between female and male BWF1 mice. Macrophage efferocytosis was deficient in female compared to male BWF1 mice but could be restored by feeding male microbiota. Further transcriptomic analysis of the genes upregulated in male BWF1 mice revealed enrichment of genes stimulated by PPARγ and LXR signaling. Our previous fecal metabolomics analyses identified metabolites in male BWF1 mice that can activate PPARγ and LXR signaling and identified one in particular, phytanic acid, that is a very potent agonist. We show here that treatment of female BWF1 splenic macrophages with phytanic acid restores efferocytic activity via activation of the PPARγ and LXR signaling pathways. Furthermore, we found phytanic acid may restore female BWF1 macrophage efferocytosis through upregulation of the proefferocytic gene CD36. Taken together, our data indicate that metabolites produced by BWF1 male microbiota can enhance macrophage efferocytosis and, through this mechanism, could potentially influence lupus progression.

Binding of hnRNP I-vRNA Regulates Sindbis Virus Structural Protein Expression to Promote Particle Infectivity.

Alphaviruses cause significant outbreaks of febrile illness and debilitating multi-joint arthritis for prolonged periods after initial infection. We have previously reported that several host hnRNP proteins bind to the Sindbis virus (SINV) RNAs, and disrupting the sites of these RNA-protein interactions results in decreased viral titers in tissue culture models of infection. Intriguingly, the primary molecular defect associated with the disruption of the hnRNP interactions is enhanced viral structural protein expression; however, the precise underlying mechanisms spurring the enhanced gene expression remain unknown. Moreover, our previous efforts were unable to functionally dissect whether the observed phenotypes were due to the loss of hnRNP binding or the incorporation of polymorphisms into the primary nucleotide sequence of SINV. To determine if the loss of hnRNP binding was the primary cause of attenuation or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to restore the binding of the hnRNP proteins in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction by incorporating the 20nt bovine immunodeficiency virus transactivation RNA response (BIV-TAR) at the site of the native hnRNP I interaction sequence, which will bind with high specificity to proteins tagged with a TAT peptide. The reestablishment of the hnRNP I-vRNA interaction via the BIV-TAR/TAT tethering approach restored the phenotype back to wild-type levels. This included an apparent decrease in structural protein expression in the absence of the native primary nucleotide sequences corresponding to the hnRNP I interaction site. Collectively, the characterization of the hnRNP I interaction site elucidated the role of hnRNPs during viral infection.

Combining Community Wastewater Genomic Surveillance with State Clinical Surveillance: A Framework for SARS-CoV-2 Public Health Practice.

This study aimed to develop a framework for combining community wastewater surveillance with state clinical surveillance for the confirmation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants within the community and to provide recommendations on how to expand on such research and apply the findings in public health responses. Wastewater samples were collected weekly from 17 geographically resolved locations in Louisville/Jefferson County, Kentucky (USA), from February 10 to December 13, 2021. Genomic surveillance and quantitative reverse transcription PCR (RT-qPCR) platforms were used to screen for SARS-CoV-2 in wastewater, and state clinical surveillance was used for confirmation. The study results highlighted an increased epidemiological value of combining community wastewater genomic surveillance and RT-qPCR with conventional case-auditing methods. The spatial scale and temporal frequency of wastewater sampling provided promising sensitivity and specificity for gaining public health screening insights about SARS-CoV-2 emergence, seeding, and spread in communities. Improved national surveillance systems are needed against future pathogens and variants, and wastewater-based genomic surveillance exhibits great potential when coupled with clinical testing. This paper presents evidence that complementary wastewater and clinical testing are cost-effectively enhanced when used in combination, as they provide a strong tool for a joint public health framework. Future pathogens of interest may be examined in either a targeted fashion or using a more global approach where all pathogens are monitored. This study has also provided novel insights developed from evidence-based public health practices.

The Detection of Periodic Reemergence Events of SARS-CoV-2 Delta Strain in Communities Dominated by Omicron.

Despite entering an endemic phase, SARS-CoV-2 remains a significant burden to public health across the global community. Wastewater sampling has consistently proven utility to understanding SARS-CoV-2 prevalence trends and genetic variation as it represents a less biased assessment of the corresponding communities. Here, we report that ongoing monitoring of SARS-CoV-2 genetic variation in samples obtained from the wastewatersheds of the city of Louisville in Jefferson county Kentucky has revealed the periodic reemergence of the Delta strain in the presence of the presumed dominant Omicron strain. Unlike previous SARS-CoV-2 waves/emergence events, the Delta reemergence events were geographically restricted in the community and failed to spread into other areas as determined by wastewater analyses. Moreover, the reemergence of the Delta strain did not correlate with vaccination rates as communities with lower relative vaccination have been, to date, not affected. Importantly, Delta reemergence events correlate with increased public health burdens, as indicated by increased daily case rates and mortality relative to non-Delta wastewatershed communities. While the underlying reasons for the reemergence of the Delta variant remain unclear, these data reaffirm the ongoing importance of wastewater genomic analyses towards understanding SARS-CoV-2 as it enters the endemic phase.

De-Coding the Contributions of the Viral RNAs to Alphaviral Pathogenesis.

Alphaviruses are positive-sense RNA arboviruses that are capable of causing severe disease in otherwise healthy individuals. There are many aspects of viral infection that determine pathogenesis and major efforts regarding the identification and characterization of virulence determinants have largely focused on the roles of the nonstructural and structural proteins. Nonetheless, the viral RNAs of the alphaviruses themselves play important roles in regard to virulence and pathogenesis. In particular, many sequences and secondary structures within the viral RNAs play an important part in the development of disease and may be considered important determinants of virulence. In this review article, we summarize the known RNA-based virulence traits and host:RNA interactions that influence alphaviral pathogenesis for each of the viral RNA species produced during infection. Overall, the viral RNAs produced during infection are important contributors to alphaviral pathogenesis and more research is needed to fully understand how each RNA species impacts the host response to infection as well as the development of disease.

Interactions between capsid and viral RNA regulate Chikungunya virus translation in a host-specific manner.

Alphaviruses are positive sense, RNA viruses commonly transmitted by an arthropod vector to a mammalian or avian host. In recent years, a number of the Alphavirus members have reemerged as public health concerns. Transmission from mosquito vector to vertebrate hosts requires an understanding of the interaction between the virus and both vertebrate and insect hosts to develop rational intervention strategies. The current study uncovers a novel role for capsid protein during Chikungunya virus replication whereby the interaction with viral RNA in the E1 coding region regulates protein synthesis processes early in infection. Studies done in both the mammalian and mosquito cells indicate that interactions between viral RNA and capsid protein have functional consequences that are host species specific. Our data support a vertebrate-specific role for capsid:vRNA interaction in temporally regulating viral translation in a manner dependent on the PI3K-AKT-mTOR pathway.

The Alphaviral Capsid Protein Inhibits IRAK1-Dependent TLR Signaling.

Alphaviruses are arthropod-borne RNA viruses which can cause either mild to severe febrile arthritis which may persist for months, or encephalitis which can lead to death or lifelong cognitive impairments. The non-assembly molecular role(s), functions, and protein-protein interactions of the alphavirus capsid proteins have been largely overlooked. Here we detail the use of a BioID2 biotin ligase system to identify the protein-protein interactions of the Sindbis virus capsid protein. These efforts led to the discovery of a series of novel host-pathogen interactions, including the identification of an interaction between the alphaviral capsid protein and the host IRAK1 protein. Importantly, this capsid-IRAK1 interaction is conserved across multiple alphavirus species, including arthritogenic alphaviruses SINV, Ross River virus, and Chikungunya virus; and encephalitic alphaviruses Eastern Equine Encephalitis virus, and Venezuelan Equine Encephalitis virus. The impact of the capsid-IRAK1 interaction was evaluated using a robust set of cellular model systems, leading to the realization that the alphaviral capsid protein specifically inhibits IRAK1-dependent signaling. This inhibition represents a means by which alphaviruses may evade innate immune detection and activation prior to viral gene expression. Altogether, these data identify novel capsid protein-protein interactions, establish the capsid-IRAK1 interaction as a common alphavirus host-pathogen interface, and delineate the molecular consequences of the capsid-IRAK1 interaction on IRAK1-dependent signaling.