Catch me if you can – the crosstalk of ZIKV and the restriction factor Tetherin

Zika virus (ZIKV) is a flavivirus that is mainly transmitted by Aedes mosquitos and normally causes mild symptoms. During the outbreak in the Americas in 2015, it was associated with more severe implications, like microcephaly in new-borns and the Gullain-Barré syndrome. The lack of specific vaccines and cures strengthen the need for a deeper understanding of the virus life cycle and virus-host interactions. The restriction factor tetherin (THN) is an interferon-inducible cellular protein with broad antiviral properties. It is known to inhibit the release of various enveloped viruses by tethering them to each other and to the cell membrane, thereby preventing their further spread. On the other hand, different viruses have developed various escape strategies against THN. Analysis of the crosstalk between ZIKV and THN revealed that in spite of a strong induction of THN mRNA expression in ZIKV-infected cells, this is not reflected by an elevated protein level of THN. Contrariwise, the THN protein level is decreased due to a reduced half-life. The increased degradation of THN in ZIKV infected cells involves the endo-lysosomal system, but does not depend on the early steps of autophagy. Enrichment of THN by depletion of the ESCRT-0 protein HRS diminishes ZIKV release and spread, which points out the capacity of THN to restrict ZIKV and explains the enhanced THN degradation in infected cells as an effective viral escape strategy. Importance Although tetherin expression is strongly induced by ZIKV infection there is a reduction in the amount of tetherin protein. This is due to an enhanced lysosomal degradation. However, if tetherin level is rescued release of ZIKV is impaired. This shows that tetherin is a restriction factor for ZIKV and the induction of an efficient degradation represents a viral escape strategy. To our knowledge this is the first study that describes and characterizes tetherin as an restriction factor for ZIKV life cycle.

Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

ABSTRACTAlphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5’UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3’ untranslated region (3’UTR) of the virus. We investigated the potential role of variability in the 3’UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3’UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3’UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3’UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3’UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

Trim28 acts as restriction factor of prototype foamy virus replication by modulating H3K9me3 marks and destabilizing the viral transactivator Tas

Background Prototype foamy virus (PFV) is nonpathogenic complex retroviruses that express a transcriptional transactivator Tas, which is essential for the activity of viral long terminal repeat (LTR) promoter and internal promoter (IP). Tripartite motif-containing protein 28 (Trim28) is well known as a scaffold protein normally enriched in gene promoter region to repress transcription. We sought to determine if whether Trim28 could be enriched in PFV promoter region to participate the establishment of PFV latency infection. Results In this study, we show that Trim28 restricts Tas-dependent transactivation activity of PFV promoter and negatively regulates PFV replication. Trim28 was found to be enriched in LTR instead of IP promoter regions of PFV genome and contribute to the maintenance of histone H3K9me3 marks on the LTR promoter. Furthermore, Trim28 interacts with Tas and colocalizes with Tas in the nucleus. Besides, we found that Trim28, an E3 ubiquitin ligase, binds directly to and promotes Tas for ubiquitination and degradation. And the RBCC domain of Trim28 is required for the ubiquitination and degradation of Tas. Conclusions Collectively, our findings not only identify a host factor Trim28 negatively inhibits PFV replication by acting as transcriptional restriction factor enriched in viral LTR promoter through modulating H3K9me3 mark here, but also reveal that Trim28 mediated ubiquitin proteasome degradation of Tas as a mechanism underlying Trim28 restricts Tas-dependent transcription activity of PFV promoter and PFV replication. These findings provide new insights into the process of PFV latency establishment. Graphical Abstract

Cryo-EM structure of the Smc5/6 holo-complex

The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral integration. Here, we report the cryo-EM structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells --providing a full overview of the complex in its apo / non-liganded form, and revealing how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the "W-loop" of Smc4 or "F-loop" of Smc1, mediates an essential interaction with Nse1. Taken together, these data confirm a degree of functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

MicroRNA-376b-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication by Targeting Viral Restriction Factor TRIM22

Porcine reproductive and respiratory syndrome virus is a major economically significant pathogen and has evolved several strategies to evade host's antiviral response and provide favorable conditions for survival. In the present study, we demonstrated that a host microRNA, miR-376b-3p, was upregulated by PRRSV infection through the viral components, nsp4 and nsp11, and miR-376b-3p can directly target tripartite motif-containing 22 (TRIM22) to impair its anti-PRRSV activity, thus facilitating the replication of PRRSV. Meanwhile, we found that TRIM22 induced degradation of the nucleocapsid protein (N) of PRRSV by interacting with N protein to inhibit PRRSV replication, and further study indicated that TRIM22 could enhance the activation of lysosomal pathway by interacting with LC3 to induce lysosomal degradation of N protein. In conclusion, PRRSV increased miR-376b-3p expression and hijacked the host miR-376b-3p to promote PRRSV replication by impairing the antiviral effect of TRIM22. Therefore, our finding outlines a novel strategy of immune evasion exerted by PRRSV, which is helpful for better understanding the pathogenesis of PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes enormous economic losses each year in the swine industry worldwide. MicroRNAs (miRNAs) play important roles during viral infections via modulating the expression of viral or host genes at post-transcriptional level. TRIM22 has recently been identified as a key restriction factor that inhibited the replication of a number of human virus such as HIV, ECMV, HCV, HBV, IAV, and RSV. Here we showed that host miR-376b-3p could be up-regulated by PRRSV and functioned to impair the anti-PRRSV role of TRIM22 to facilitate PRRSV replication. Meanwhile, we found that TRIM22 inhibited the replication of PRRSV by interacting with viral N protein and accelerating its degradation through the lysosomal pathway. Collectively, the paper described a novel mechanism that PRRSV exploited the host miR-376b-3p to evade antiviral responses and provided a new insight into the study of virus-host interactions.

Host restriction factor A3G inhibits the replication of Enterovirus D68 through competitively binding 5’ UTR with PCBP1

The host restriction factor APOBEC3G (A3G) presents extensively inhibition on a variety of viruses, including retroviruses, DNA and RNA viruses. Our recent study showed that A3G inhibits enterovirus 71 (EV71) and coxsackievirus A16 (CA16) via competitively binding 5’UTR with the host protein poly(C)-binding protein 1 (PCBP1) that is required for multiple EVs replication. However, in addition to EV71 and CA16, whether A3G inhibits other EVs has not been investigated. Here, we demonstrate that A3G could inhibit EVD68 replication, which needs PCBP1 for its replication, but not CA6 that PCBP1 is dispensable for CA6 replication. Further investigation revealed that nucleic acid binding activity of A3G is required for EVD68 restriction, which is similar to the mechanism presented in EV71 restriction. Mechanistically, A3G competitively binds to the cloverleaf (1–123) and the stem-loop IV (234-446) domains of EVD68 5’UTR with PCBP1, thereby inhibiting the 5'UTR activity of EVD68, whereas A3G doesn’t interact with CA6 5’UTR results in no effect on CA6 replication. Moreover, non-structural protein 2C encoded by EVD68 overcomes A3G suppression through inducing A3G degradation via the autophagy-lysosome pathway. Our finding revealed that A3G might have broad spectrum antiviral activity against multiple EVs through the general mechanism, which might provide important information for the development of anti-EVs strategy. Importance As the two major pathogens causing hand, food, and mouth disease (HFMD), EV71 and CA16 attract more attention for the discovery of pathogenesis, the involvement of cellular proteins and so on. However, other EVs such as CA6 or EVD68 constantly occurred sporadic or might spread widely in recent years worldwide. Therefore, more information related to these EVs needs to be further investigated so as to develop broad-spectrum anti-EVs inhibitor. In this study, we first reveal that PCBP1 involved in PV and EV71 virus replication, also is required for the replication of EVD68 but not CA6. Then we found that the host restriction factor A3G specifically inhibits the replication of EVD68 but not CA6 via competitively binding to the 5’UTR of EVD68 with PCBP1. Our findings broaden the knowledge related to EVs replication and the interplay between EVs and host factors.

Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor

Interferons establish an antiviral state in responding cells through the induction of hundreds of interferon-stimulated genes (ISGs). ISGs antagonize viral pathogens directly through diverse mechanisms acting at different stages of viral life cycles, and indirectly by modulating cell cycle and promoting programmed cell death. The mechanisms of action and viral specificities for most ISGs remain incompletely understood. To enable the high throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating the potentially confounding effects of endogenous IFN and potential antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG induction in isogenic cell lines with and without the capacity to respond to IFN. Engineered CRISPRa cell lines demonstrated inducible, robust, and specific gRNA-directed expression of ISGs, which are functional in restricting viral infection. Using this platform, we screened for ISGs that restrict SARS-CoV-2, the causative agent of the COVID-19 pandemic. Results included ISGs previously described to restrict SARS-CoV-2 as well as multiple novel candidate antiviral factors. We validated a subset of candidate hits by complementary targeted CRISPRa and ectopic cDNA expression infection experiments, which, among other hits, confirmed OAS1 as a SARS-CoV-2 restriction factor. OAS1 exhibited strong antiviral effects against SARS-CoV-2, and these effects required OAS1 catalytic activity. These studies demonstrate a robust, high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by the identification of multiple novel SARS-CoV-2 restriction factors.