Exploring the diversity and evolution of tombus-like viruses: phylogenetic analysis, recombination events, and suppressor protein homologs.

This study focuses on the phylogenetic analysis of previously unclassified tombus-like viruses, which are characterized by the presence of homologs of the suppressor protein p19. The primary objectives of this research were to investigate the evolutionary relationships among these viruses and to explore the impact of suppressor proteins and recombination events on their evolution. A dataset comprising 94 viral sequences was analyzed to achieve these goals. The phylogenetic analysis revealed the presence of two distinct clusters within the tombus-like virus group. One cluster consisted of viruses that encoded p19-like RNA suppressors, while the other cluster comprised viruses encoding p14-like suppressors. Based on these findings, we propose the classification of PGT-pt108 as an isolate of carnation Italian ringspot virus (CIRV), and both Tombusviridae sp. s48-k141_139792 and Tombusviridae sp. s51-k141_185213 as isolates of tomato bushy stunt virus (TBSV). Furthermore, this study suggests the establishment of two new genera within the family Tombusviridae, based on the observed divergence and distinct characteristics of these tombus-like viruses. Through the analysis of recombination events, we provide insights into the interspecies movement of CIRV, which is reflected in its phylogenetic positioning. This research contributes to our understanding of the evolutionary dynamics and classification of tombus-like viruses, shedding light on the role of suppressor proteins and recombination events in their evolution and interspecies transmission.

The complete genome sequence of Neckar River virus confirms it to be a distinct member of the genus Tombusvirus in the family Tombusviridae.

Neckar River virus (NRV), first isolated from a water sample of the Neckar River (Germany) in the 1980s, was serologically characterized as a novel tombusvirus. In this study, the complete genome sequence was determined, and an infectious full-length cDNA clone was constructed. The genome organization of NRV (DSMZ PV-0270) resembles that of tombusviruses. The genome consists of 4739 nucleotides and contains five open reading frames (ORFs) and one additional putative ORF (pX) in the 3'-terminal region. Phylogenetic analysis and sequence comparisons confirmed NRV to be a member of the species Tombusvirus neckarfluminis in the genus Tombusvirus. The infectious full-length cDNA clone was constructed using Gibson assembly and subsequent infection of Nicotiana benthamiana plants by Rhizobium radiobacter inoculation. The virus derived from the full-length cDNA clone caused symptoms resembling those caused by the wild-type virus, but slightly milder.

The Quest to Identify a New Virus Disease of Sunflower from Nebraska.

Between 2010 and 2018, sunflower plants exhibiting virus-like symptoms, including stunting, mottling, and chlorotic ringspots on leaves, were observed from commercial fields and research plots from four sites within three distinct counties of western Nebraska (Box Butte, Kimball, and Scotts Bluff). Near identical symptoms from field samples were reproduced on seedlings mechanically in the greenhouse on multiple occasions, confirming the presence of a sap-transmissible virus from each site. Symptomatic greenhouse-inoculated plants from the 2010 and 2011 Box Butte samples tested negative for sunflower mosaic virus (SuMV), sunflower chlorotic mottle virus (SuCMoV), and all potyviruses in general by ELISA and RT-PCR. Similar viral-like symptoms were later observed on plants in a commercial sunflower field in Kimball County in 2014, and again from volunteers in research plots in Scotts Bluff County in 2018. Samples from both of these years were again successfully reproduced on seedlings in the greenhouse as before following mechanical transmissions. Symptom expression for all years began 12 to 14 days after inoculation as mild yellow spots followed by the formation of chlorotic ringspots from the mottled pattern. The culture from 2014 tested negatively for three groups of nepoviruses via RT-PCR, ruling this group out. However, transmission electron microscopy assays of greenhouse-infected plants from both 2014 and 2018 revealed the presence of distinct, polyhedral virus particles. With the use of high throughput sequencing and RT-PCR, it was confirmed that the infections from both years were caused by a new virus in the tombusvirus genus and was proposed to be called Sunflower ring spot mottle virus (SuRSMV). Although the major objective of this project was to identify the causal agent of the disease, it became evident that the diagnostic journey itself, with all the barriers encountered on the 10-year trek, was actually more important and impactful than identification.

Multifunctional role of the co-opted Cdc48 AAA+ ATPase in tombusvirus replication.

Replication of positive-strand RNA viruses depends on usurped cellular membranes and co-opted host proteins. Based on pharmacological inhibition and genetic and biochemical approaches, the authors identified critical roles of the cellular Cdc48 unfoldase/segregase protein in facilitating the replication of tomato bushy stunt virus (TBSV). We show that TBSV infection induces the expression of Cdc48 in Nicotiana benthamiana plants. Cdc48 binds to the TBSV replication proteins through its N-terminal region. In vitro TBSV replicase reconstitution experiments demonstrated that Cdc48 is needed for efficient replicase assembly and activity. Surprisingly, the in vitro replication experiments also showed that excess amount of Cdc48 facilitates the disassembly of the membrane-bound viral replicase-RNA template complex. Cdc48 is also needed for the recruitment of additional host proteins. Because several human viruses, including flaviviruses, utilize Cdc48, also called VCP/p97, for replication, we suggest that Cdc48 might be a common panviral host factor for plant and animal RNA viruses.

The centromeric histone CenH3 is recruited into the tombusvirus replication organelles.

Tombusviruses, similar to other (+)RNA viruses, exploit the host cells by co-opting numerous host components and rewiring cellular pathways to build extensive virus-induced replication organelles (VROs) in the cytosol of the infected cells. Most molecular resources are suboptimal in susceptible cells and therefore, tomato bushy stunt virus (TBSV) drives intensive remodeling and subversion of many cellular processes. The authors discovered that the nuclear centromeric CenH3 histone variant (Cse4p in yeast, CENP-A in humans) plays a major role in tombusvirus replication in plants and in the yeast model host. We find that over-expression of CenH3 greatly interferes with tombusvirus replication, whereas mutation or knockdown of CenH3 enhances TBSV replication in yeast and plants. CenH3 binds to the viral RNA and acts as an RNA chaperone. Although these data support a restriction role of CenH3 in tombusvirus replication, we demonstrate that by partially sequestering CenH3 into VROs, TBSV indirectly alters selective gene expression of the host, leading to more abundant protein pool. This in turn helps TBSV to subvert pro-viral host factors into replication. We show this through the example of hypoxia factors, glycolytic and fermentation enzymes, which are exploited more efficiently by tombusviruses to produce abundant ATP locally within the VROs in infected cells. Altogether, we propose that subversion of CenH3/Cse4p from the nucleus into cytosolic VROs facilitates transcriptional changes in the cells, which ultimately leads to more efficient ATP generation in situ within VROs by the co-opted glycolytic enzymes to support the energy requirement of virus replication. In summary, CenH3 plays both pro-viral and restriction functions during tombusvirus replication. This is a surprising novel role for a nuclear histone variant in cytosolic RNA virus replication.

Key tethering function of Atg11 autophagy scaffold protein in formation of virus-induced membrane contact sites during tombusvirus replication.

Positive-strand RNA viruses induce the biogenesis of viral replication organelles (VROs), which support viral replication in infected cells. VRO formation requires viral replication proteins, co-opted host factors and intracellular membranes. Here, we show that the conserved Atg11 autophagy scaffold protein is co-opted by Tomato bushy stunt virus (TBSV) via direct interactions with the viral replication proteins. Deletion of ATG11 in yeast or knockdown of the homologous Atg11 in plants led to reduced tombusvirus replication, thus indicating pro-viral function for Atg11. Based on co-purification, BiFC and proximity-labeling experiments, we find that Atg11 is co-opted to stabilize virus-induced membrane contact sites (vMCS) within VROs. We propose that the tethering and scaffold function of Atg11 is critical in vMCSs for lipid enrichment. Absence of Atg11 interferes with sterols enrichment in VROs, rendering VROs RNAi-sensitive. Altogether, the expanding roles of co-opted host proteins with tethering functions suggest that the tombusvirus VROs are elaborate structures.

Race against Time between the Virus and Host: Actin-Assisted Rapid Biogenesis of Replication Organelles is Used by TBSV to Limit the Recruitment of Cellular Restriction Factors.

Positive-strand RNA viruses build large viral replication organelles (VROs) with the help of coopted host factors. Previous works on tomato bushy stunt virus (TBSV) showed that the p33 replication protein subverts the actin cytoskeleton by sequestering the actin depolymerization factor, cofilin, to reduce actin filament disassembly and stabilize the actin filaments. Then, TBSV utilizes the stable actin filaments as "trafficking highways" to deliver proviral host factors into the protective VROs. In this work, we show that the cellular intrinsic restriction factors (CIRFs) also use the actin network to reach VROs and inhibit viral replication. Disruption of the actin filaments by expression of the Legionella RavK protease inhibited the recruitment of plant CIRFs, including the CypA-like Roc1 and Roc2 cyclophilins, and the antiviral DDX17-like RH30 DEAD box helicase into VROs. Conversely, temperature-sensitive actin and cofilin mutant yeasts with stabilized actin filaments reduced the levels of copurified CIRFs, including cyclophilins Cpr1, CypA, Cyp40-like Cpr7, cochaperones Sgt2, the Hop-like Sti1, and the RH30 helicase in viral replicase preparations. Dependence of the recruitment of both proviral and antiviral host factors into VROs on the actin network suggests that there is a race going on between TBSV and its host to exploit the actin network and ultimately to gain the upper hand during infection. We propose that, in the highly susceptible plants, tombusviruses efficiently subvert the actin network for rapid delivery of proviral host factors into VROs and ultimately overcome host restriction factors via winning the recruitment race and overwhelming cellular defenses. IMPORTANCE Replication of positive-strand RNA viruses is affected by the recruitment of host components, which provide either proviral or antiviral functions during virus invasion of infected cells. The delivery of these host factors into the viral replication organelles (VROs), which represent the sites of viral RNA replication, depends on the cellular actin network. Using TBSV, we uncover a race between the virus and its host with the actin network as the central player. We find that in susceptible plants, tombusviruses exploit the actin network for rapid delivery of proviral host factors into VROs and ultimately overcome host restriction factors. In summary, this work demonstrates that the actin network plays a major role in determining the outcome of viral infections in plants.

RNA and Protein Determinants Mediate Differential Binding of miRNAs by a Viral Suppressor of RNA Silencing Thus Modulating Antiviral Immune Responses in Plants.

Many plant viruses express suppressor proteins (VSRs) that can inhibit RNA silencing, a central component of antiviral plant immunity. The most common activity of VSRs is the high-affinity binding of virus-derived siRNAs and thus their sequestration from the silencing process. Since siRNAs share large homologies with miRNAs, VSRs like the Tombusvirus p19 may also bind miRNAs and in this way modulate cellular gene expression at the post-transcriptional level. Interestingly, the binding affinity of p19 varies considerably between different miRNAs, and the molecular determinants affecting this property have not yet been adequately characterized. Addressing this, we analyzed the binding of p19 to the miRNAs 162 and 168, which regulate the expression of the important RNA silencing constituents Dicer-like 1 (DCL1) and Argonaute 1 (AGO1), respectively. p19 binds miRNA162 with similar high affinity as siRNA, whereas the affinity for miRNA168 is significantly lower. We show that specific molecular features, such as mismatches and 'G-U wobbles' on the RNA side and defined amino acid residues on the VSR side, mediate this property. Our observations highlight the remarkable adaptation of VSR binding affinities to achieve differential effects on host miRNA activities. Moreover, they show that even minimal changes, i.e., a single base pair in a miRNA duplex, can have significant effects on the efficiency of the plant antiviral immune response.

Viral suppressor of RNA silencing in vascular plants also interferes with the development of the bryophyte Physcomitrella patens.

Plant viruses are important pathogens able to overcome plant defense mechanisms using their viral suppressors of RNA silencing (VSR). Small RNA pathways of bryophytes and vascular plants have significant similarities, but little is known about how viruses interact with mosses. This study elucidated the responses of Physcomitrella patens to two different VSRs. We transformed P. patens plants to express VSR P19 from tomato bushy stunt virus and VSR 2b from cucumber mosaic virus, respectively. RNA sequencing and quantitative PCR were used to detect the effects of VSRs on gene expression. Small RNA (sRNA) sequencing was used to estimate the influences of VSRs on the sRNA pool of P. patens. Expression of either VSR-encoding gene caused developmental disorders in P. patens. The transcripts of four different transcription factors (AP2/erf, EREB-11 and two MYBs) accumulated in the P19 lines. sRNA sequencing revealed that VSR P19 significantly changed the microRNA pool in P. patens. Our results suggest that VSR P19 is functional in P. patens and affects the abundance of specific microRNAs interfering with gene expression. The results open new opportunities for using Physcomitrella as an alternative system to study plant-virus interactions.

A plant-infecting subviral RNA associated with poleroviruses produces a subgenomic RNA which resists exonuclease XRN1 in vitro.

Subviral agents are nucleic acids which lack the features for classification as a virus. Tombusvirus-like associated RNAs (tlaRNAs) are subviral positive-sense, single-stranded RNAs that replicate autonomously, yet depend on a coinfecting virus for encapsidation and transmission. TlaRNAs produce abundant subgenomic RNA (sgRNA) upon infection. Here, we investigate how the well-studied tlaRNA, ST9, produces sgRNA and its function. We found ST9 is a noncoding RNA, due to its lack of protein coding capacity. We used resistance assays with eukaryotic Exoribonuclease-1 (XRN1) to investigate sgRNA production via incomplete degradation of genomic RNA. The ST9 3' untranslated region stalled XRN1 very near the 5' sgRNA end. Thus, the XRN family of enzymes drives sgRNA accumulation in ST9-infected tissue by incomplete degradation of ST9 RNA. This work suggests tlaRNAs are not just parasites of viruses with compatible capsids, but also mutually beneficial partners that influence host cell RNA biology.

RNA Structure Protects the 5' End of an Uncapped Tombusvirus RNA Genome from Xrn Digestion.

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5' termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5'-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5'-to-3' exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5'-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5' access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5' terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5' access, we have termed this 5'-terminal structure an Xrn-evading RNA, or xeRNA. These and other findings demonstrate that the 5'UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5' terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5' capped. Here, we explored how a species in the type genus Tombusvirus protects its genomic 5' end from cellular nuclease attack. Our results revealed that the 5'-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5'-to-3' exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5'-triphosphorylated RNA termini into 5'-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5'-end-attacking cellular enzymes.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Infectious in vitro transcripts from a cDNA clone of a Japanese gentian isolate of Sikte waterborne virus, which shows host-specific low-temperature-dependent replication.

Tombusviruses have been identified in several crops, including gentian virus A (GeVA) in Japanese gentian. In this study, we isolated another tombusvirus, Sikte waterborne virus strain C1 (SWBV-C1), from Japanese gentian. Although SWBV-C1 and GeVA are not closely related, SWBV-C1, like GeVA, showed host-specific low-temperature-dependent replication in gentian and arabidopsis. The use of in vitro transcripts from full-length cDNA clones of SWBV-C1 genomic RNA as inocula confirmed these properties, indicating that the identified genomic RNA sequences encode viral factors responsible for the characteristic features of SWBV-C1.

Tombusviruses orchestrate the host endomembrane system to create elaborate membranous replication organelles.

Positive-strand RNA viruses depend on intensive manipulation of subcellular organelles and membranes to create unique viral replication organelles (VROs), which represent the sites of robust virus replication. The host endomembrane-based protein-trafficking and vesicle-trafficking pathways are specifically targeted by many (+)RNA viruses to take advantage of their rich resources. We summarize the critical roles of co-opted endoplasmic reticulum subdomains and associated host proteins and COPII vesicles play in tombusvirus replication. We also present the surprising contribution of the early endosome and the retromer tubular transport carriers to VRO biogenesis. The central player is tomato bushy stunt virus (TBSV), which provides an outstanding system based on the identification of a complex network of interactions with the host cells. We present the emerging theme on how TBSV uses tethering and membrane-shaping proteins and lipid modifying enzymes to build the sophisticated VRO membranes with unique lipid composition.

Co-opting of nonATP-generating glycolytic enzymes for TBSV replication.

Positive-strand RNA viruses build viral replication organelles (VROs) with the help of co-opted host factors. The energy requirement of intensive viral replication processes is less understood. Previous studies on tomato bushy stunt virus (TBSV) showed that tombusviruses hijack two ATP-producing glycolytic enzymes to produce ATP locally within VROs. In this work, we performed a cDNA library screen with Arabidopsis thaliana proteins and the TBSV p33 replication protein. The p33 - plant interactome contained highly conserved glycolytic proteins. We find that the glycolytic Hxk2 hexokinase, Eno2 phosphopyruvate hydratase and Fba1 fructose 1,6-bisphosphate aldolase are critical for TBSV replication in yeast or in a cell-free replicase reconstitution assay. The recruitment of Fba1 is important for the local production of ATP within VROs. Altogether, our data support the model that TBSV recruits and compartmentalizes possibly most members of the glycolytic pathway. This might allow TBSV to avoid competition with the host for ATP.

Targeting of cucumber necrosis virus coat protein to the chloroplast stroma attenuates host defense response.

Cucumber necrosis virus (CNV) is a (+)ssRNA virus that elicits spreading local and systemic necrosis in Nicotiana benthamiana. We previously showed that the CNV coat protein (CP) arm functions as a chloroplast transit peptide that targets a CP fragment containing the S and P domains to chloroplasts during infection. Here we show that several CP arm mutants that inefficiently target chloroplasts, along with a mutant that lacks the S and P domains, show an early onset of more localized necrosis along with protracted induction of pathogenesis related protein (PR1a). Agroinfiltrated CNV CP is shown to interfere with CNV p33 and Tomato bushy stunt virus p19 induced necrosis. Additionally, we provide evidence that a CP mutant that does not detectably enter the chloroplast stroma induces relatively higher levels of several plant defense-related genes compared to WT CNV. Together, our data suggest that targeting of CNV CP to the chloroplast stroma interferes with chloroplast-mediated plant defense.

Complete genome sequence of Sikte (Sitke) waterborne virus, a member of the genus Tombusvirus.

The 4704-nt genome sequence of Sikte waterborne virus (SWV), determined by fragmented and primer ligated dsRNA sequencing and by direct Sanger sequencing, is linear, nonsegmented and has the five ORFs of other tombusviruses. The 5' and 3' untranslated regions (UTRs) are 150 and 335 nt long, respectively. Phylogenetic analysis of the coat protein revealed that SWV is related to CymRSV and PNSV, but that of the SWV replicase protein, the p92 readthrough protein, indicated a close relationship to CNV. These phylogenetic analyses suggest the occurrence of recombination events in SWV, as reported previously for other tombusviruses.

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles.

Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.

Key interplay between the co-opted sorting nexin-BAR proteins and PI3P phosphoinositide in the formation of the tombusvirus replicase.

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.