Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells.

Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.

Circulating Immune Cells Mediate a Systemic RNAi-Based Adaptive Antiviral Response in Drosophila.

Effective antiviral protection in multicellular organisms relies on both cell-autonomous and systemic immunity. Systemic immunity mediates the spread of antiviral signals from infection sites to distant uninfected tissues. In arthropods, RNA interference (RNAi) is responsible for antiviral defense. Here, we show that flies have a sophisticated systemic RNAi-based immunity mediated by macrophage-like haemocytes. Haemocytes take up dsRNA from infected cells and, through endogenous transposon reverse transcriptases, produce virus-derived complementary DNAs (vDNA). These vDNAs template de novo synthesis of secondary viral siRNAs (vsRNA), which are secreted in exosome-like vesicles. Strikingly, exosomes containing vsRNAs, purified from haemolymph of infected flies, confer passive protection against virus challenge in naive animals. Thus, similar to vertebrates, insects use immune cells to generate immunological memory in the form of stable vDNAs that generate systemic immunity, which is mediated by the vsRNA-containing exosomes.

RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.

Fanconi Anemia Proteins Function in Mitophagy and Immunity.

Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.

VLDLR and ApoER2 are receptors for multiple alphaviruses.

Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1-3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2-E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD-Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2-E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD-Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.

System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection.

The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

Extreme infectious titer variability in individual Aedes aegypti mosquitoes infected with Sindbis virus is associated with both differences in virus population structure and dramatic disparities in specific infectivity.

Variability in how individuals respond to pathogens is a hallmark of infectious disease, yet the basis for individual variation in host response is often poorly understood. The titer of infectious virus among individual mosquitoes infected with arboviruses is frequently observed to vary by several orders of magnitude in a single experiment, even when the mosquitoes are highly inbred. To better understand the basis for this titer variation, we sequenced populations of Sindbis virus (SINV) obtained from individual infected Aedes aegypti mosquitoes that, despite being from a highly inbred laboratory colony, differed in their titers of infectious virus by approximately 10,000-fold. We observed genetic differences between these virus populations that indicated the virus present in the midguts of low titer mosquitoes was less fit than that of high titer mosquitoes, possibly due to founder effects that occurred during midgut infection. Furthermore, we found dramatic differences in the specific infectivity or SI (the ratio of infectious units/viral genome equivalents) between these virus populations, with the SI of low titer mosquitoes being up to 10,000-fold lower than that of high titer mosquitoes. Despite having similar amounts of viral genomes, low titer mosquitoes appeared to contain less viral particles, suggesting that viral genomes were packaged into virions less efficiently than in high titer mosquitoes. Finally, antibiotic treatment, which has been shown to suppress mosquito antiviral immunity, caused an increase in SI. Our results indicate that the extreme variation that is observed in SINV infectious titer between individual Ae. aegypti mosquitoes is due to both genetic differences between virus populations and to differences in the proportion of genomes that are packaged into infectious particles.

Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti.

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in mosquitoes.

Proteomics-based determination of double-stranded RNA interactome reveals known and new factors involved in Sindbis virus infection.

Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double-stranded (ds)RNA is a common viral by-product originating during RNA virus replication and is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated with viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by mass spectrometry analysis to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human cells. Among the identified proteins, we characterized SFPQ (splicing factor, proline-glutamine rich) as a new dsRNA-associated proviral factor upon SINV infection. We showed that SFPQ depletion reduces SINV infection in human HCT116 and SK-N-BE(2) cells, suggesting that SFPQ enhances viral production. We demonstrated that the cytoplasmic fraction of SFPQ partially colocalizes with dsRNA upon SINV infection. In agreement, we proved by RNA-IP that SFPQ can bind dsRNA and viral RNA. Furthermore, we showed that overexpression of a wild-type, but not an RNA binding mutant SFPQ, increased viral infection, suggesting that RNA binding is essential for its positive effect on the virus. Overall, this study provides the community with a compendium of dsRNA-associated factors during viral infection and identifies SFPQ as a new proviral dsRNA binding protein.

N-acetyltransferase 10 regulates alphavirus replication via N4-acetylcytidine (ac4C) modification of the lymphocyte antigen six family member E (LY6E) mRNA.

Epitranscriptomic RNA modifications can regulate the stability of mRNA and affect cellular and viral RNA functions. The N4-acetylcytidine (ac4C) modification in the RNA viral genome was recently found to promote viral replication; however, the mechanism by which RNA acetylation in the host mRNA regulates viral replication remains unclear. To help elucidate this mechanism, the roles of N-acetyltransferase 10 (NAT10) and ac4C during the infection and replication processes of the alphavirus, Sindbis virus (SINV), were investigated. Cellular NAT10 was upregulated, and ac4C modifications were promoted after alphavirus infection, while the loss of NAT10 or inhibition of its N-acetyltransferase activity reduced alphavirus replication. The NAT10 enhanced alphavirus replication as it helped to maintain the stability of lymphocyte antigen six family member E mRNA, which is a multifunctional interferon-stimulated gene that promotes alphavirus replication. The ac4C modification was thus found to have a non-conventional role in the virus life cycle through regulating host mRNA stability instead of viral mRNA, and its inhibition could be a potential target in the development of new alphavirus antivirals.IMPORTANCEThe role of N4-acetylcytidine (ac4C) modification in host mRNA and virus replication is not yet fully understood. In this study, the role of ac4C in the regulation of Sindbis virus (SINV), a prototype alphavirus infection, was investigated. SINV infection results in increased levels of N-acetyltransferase 10 (NAT10) and increases the ac4C modification level of cellular RNA. The NAT10 was found to positively regulate SINV infection in an N-acetyltransferase activity-dependent manner. Mechanistically, the NAT10 modifies lymphocyte antigen six family member E (LY6E) mRNA-the ac4C modification site within the 3'-untranslated region (UTR) of LY6E mRNA, which is essential for its translation and stability. The findings of this study demonstrate that NAT10 regulated mRNA stability and translation efficiency not only through the 5'-UTR or coding sequence but also via the 3'-UTR region. The ac4C modification of host mRNA stability instead of viral mRNA impacting the viral life cycle was thus identified, indicating that the inhibition of ac4C could be a potential target when developing alphavirus antivirals.

Replication of alphaviruses requires a pseudoknot that involves the poly(A) tail.

Alphaviruses, such as the Sindbis virus and the Chikungunya virus, are RNA viruses with a positive sense single-stranded RNA genome that infect various vertebrates, including humans. A conserved sequence element (CSE) of ∼19 nt in the 3' noncoding region is important for replication. Despite extensive mutational analysis of the CSE, no comprehensive model of this element exists to date. Here, it is shown that the CSE can form an RNA pseudoknot with part of the poly(A) tail and is similar to the human telomerase pseudoknot with which it shares 17 nt. Mutants that alter the stability of the pseudoknot were investigated in the context of a replicon of the Sindbis virus and by native gel electrophoresis. These studies reveal that the pseudoknot is required for virus replication and is stabilized by UAU base triples. The new model is discussed in relation to previous data on Sindbis virus mutants and revertants lacking (part of) the CSE.

Interferon regulatory factor 7 modulates virus clearance and immune responses to alphavirus encephalomyelitis.

IMPORTANCE: Viral encephalomyelitis outcome is dependent on host responses to neuronal infection. Interferon (IFN) is an important component of the innate response, and IFN regulatory factor (IRF) 7 is an inducible transcription factor for the synthesis of IFN-α. IRF7-deficient mice develop fatal paralysis after CNS infection with Sindbis virus, while wild-type mice recover. Irf7 -/- mice produce low levels of IFN-α but high levels of IFN-ß with induction of IFN-stimulated genes, so the reason for this difference is not understood. The current study shows that Irf7 -/- mice developed inflammation earlier but failed to clear virus from motor neuron-rich regions of the brainstem and spinal cord. Levels of IFN-γ and virus-specific antibody were comparable, indicating that IRF7 deficiency does not impair expression of these known viral clearance factors. Therefore, IRF7 is either necessary for the neuronal response to currently identified mediators of clearance or enables the production of additional antiviral factor(s) needed for clearance.

Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.

Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding.

A novel three-plasmid packaging system for chimeric SFV/SIN VRPs derived from Semliki Forest virus and Sindbis virus as a candidate gene delivery vector.

Semliki Forest virus (SFV) viral replicon particles (VRPs) have been frequently used in various animal models and clinical trials. Chimeric replicon particles offer different advantages because of their unique biological properties. We here constructed a novel three-plasmid packaging system for chimeric SFV/SIN VRPs. The capsid and envelope of SIN structural proteins were generated using two-helper plasmids separately, and the SFV replicon contained the SFV replicase gene, packaging signal of SIN, subgenomic promoter followed by the exogenous gene, and 3' UTR of SIN. The chimeric VRPs carried luciferase or eGFP as reporter genes. The fluorescence and electron microscopy results revealed that chimeric VRPs were successfully packaged. The yield of the purified chimeric VRPs was approximately 2.5 times that of the SFV VRPs (1.38 × 107 TU/ml vs. 5.41 × 106 TU/ml) (p < 0.01). Furthermore, chimeric VRPs could be stored stably at 4°C for at least 60 days. Animal experiments revealed that mice immunized with chimeric VRPs (luciferase) had stronger luciferase expression than those immunized with equivalent amount of SFV VRPs (luciferase) (p < 0.01), and successfully expressed luciferase for approximately 12 days. Additionally, the chimeric VRPs expressed the RBD of SARS-CoV-2 efficiently and induced robust RBD-specific antibody responses in mice. In conclusion, the chimeric VRPs constructed here met the requirements of a gene delivery tool for vaccine development and cancer therapy.

An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies.

DEAD box RNA helicase 5 is a new pro-viral host factor for Sindbis virus infection.

BACKGROUND: RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection. METHODS: We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments. RESULTS: In this study we demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV. CONCLUSIONS: These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.

Oncolytic Activity of Sindbis Virus with the Help of GM-CSF in Hepatocellular Carcinoma.

Hepatocellular carcinoma is a refractory tumor with poor prognosis and high mortality. Many oncolytic viruses are currently being investigated for the treatment of hepatocellular carcinoma. Based on previous studies, we constructed a recombinant GM-CSF-carrying Sindbis virus, named SINV-GM-CSF, which contains a mutation (G to S) at amino acid 285 in the nsp1 protein of the viral vector. The potential of this mutated vector for liver cancer therapy was verified at the cellular level and in vivo, respectively, and the changes in the tumor microenvironment after treatment were also described. The results showed that the Sindbis virus could effectively infect hepatocellular carcinoma cell lines and induce cell death. Furthermore, the addition of GM-CSF enhanced the tumor-killing effect of the Sindbis virus and increased the number of immune cells in the intra-tumor microenvironment during the treatment. In particular, SINV-GM-CSF was able to efficiently kill tumors in a mouse tumor model of hepatocellular carcinoma by regulating the elevation of M1-type macrophages (which have a tumor-resistant ability) and the decrease in M2-type macrophages (which have a tumor-promoting capacity). Overall, SINV-GM-CSF is an attractive vector platform with clinical potential for use as a safe and effective oncolytic virus.

Sindbis Virus Vaccine Platform: A Promising Oncolytic Virus-Mediated Approach for Ovarian Cancer Treatment.

This review article provides a comprehensive overview of a novel Sindbis virus vaccine platform as potential immunotherapy for ovarian cancer patients. Ovarian cancer is the most lethal of all gynecological malignancies. The majority of high-grade serous ovarian cancer (HGSOC) patients are diagnosed with advanced disease. Current treatment options are very aggressive and limited, resulting in tumor recurrences and 50-60% patient mortality within 5 years. The unique properties of armed oncolytic Sindbis virus vectors (SV) in vivo have garnered significant interest in recent years to potently target and treat ovarian cancer. We discuss the molecular biology of Sindbis virus, its mechanisms of action against ovarian cancer cells, preclinical in vivo studies, and future perspectives. The potential of Sindbis virus-based therapies for ovarian cancer treatment holds great promise and warrants further investigation. Investigations using other oncolytic viruses in preclinical studies and clinical trials are also presented.

Sindbis Macrodomain Poly-ADP-Ribose Hydrolase Activity Is Important for Viral RNA Synthesis.

ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.