A bivalent Adenovirus-Vectored Vaccine induces a robust humoral response, but does not protect cynomolgus macaques against a lethal challenge with Sudan virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific IgG responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no difference in survival outcomes were measured among all three groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

Pteropus vampyrus TRIM40 Is an Interferon-Stimulated Gene That Antagonizes RIG-I-like Receptors.

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats' tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNß, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

Ebola Virus VP35 Interacts Non-Covalently with Ubiquitin Chains to Promote Viral Replication Creating New Therapeutic Opportunities.

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the co-factor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity that correlated with reduced replication of infectious EBOV. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.

Lighting the way to host-directed immunotherapeutics.

Developing broad-spectrum, host-directed antiviral therapeutics can be adapted to combat emerging viruses. In this issue of Cell Chemical Biology, Maarifi and colleagues implement a Nano luciferase reporter-based protein complementation assay to screen for small molecules and identify Gilteritinib, which enhances interferon induction and antagonizes virus replication.

Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly.

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.

The RNA helicase DHX16 recognizes specific viral RNA to trigger RIG-I-dependent innate antiviral immunity.

Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.

The Role of the Host Ubiquitin System in Promoting Replication of Emergent Viruses.

Ubiquitination of proteins is a post-translational modification process with many different cellular functions, including protein stability, immune signaling, antiviral functions and virus replication. While ubiquitination of viral proteins can be used by the host as a defense mechanism by destroying the incoming pathogen, viruses have adapted to take advantage of this cellular process. The ubiquitin system can be hijacked by viruses to enhance various steps of the replication cycle and increase pathogenesis. Emerging viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses like Zika and dengue, as well as highly pathogenic viruses like Ebola and Nipah, have the ability to directly use the ubiquitination process to enhance their viral-replication cycle, and evade immune responses. Some of these mechanisms are conserved among different virus families, especially early during virus entry, providing an opportunity to develop broad-spectrum antivirals. Here, we discuss the mechanisms used by emergent viruses to exploit the host ubiquitin system, with the main focus on the role of ubiquitin in enhancing virus replication.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

TRIM Proteins in Host Defense and Viral Pathogenesis.

PURPOSE OF REVIEW: Tripartite motif (TRIM) proteins are a large group of E3 ubiquitin ligases involved in different cellular functions. Of special interest are their roles in innate immunity, inflammation, and virus replication. We discuss novel roles of TRIM proteins during virus infections that lead to increased pathogenicity. RECENT FINDINGS: TRIM proteins regulate different antiviral and inflammatory signaling pathways, mostly by promoting ubiquitination of important factors including pattern recognition receptors, adaptor proteins, kinases, and transcription factors that are involved in type I interferon and NF-κB pathways. Therefore, viruses have developed mechanisms to target TRIMs for immune evasion. New evidence is emerging indicating that viruses have the ability to directly use TRIMs and the ubiquitination process to enhance the viral replication cycle and cause increased pathogenesis. A new report on TRIM7 also highlights the potential pro-viral role of TRIMs via ubiquitination of viral proteins and suggests a novel mechanism by which ubiquitination of virus envelope protein may provide determinants of tissue and species tropism. SUMMARY: TRIM proteins have important functions in promoting host defense against virus infection; however, viruses have adapted to evade TRIM-mediated immune responses and can hijack TRIMs to ultimately increase virus pathogenesis. Only by understanding specific TRIM-virus interactions and by using more in vivo approaches can we learn how to harness TRIM function to develop therapeutic approaches to reduce virus pathogenesis.

Envelope protein ubiquitination drives entry and pathogenesis of Zika virus.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.

VAMP8 Contributes to the TRIM6-Mediated Type I Interferon Antiviral Response during West Nile Virus Infection.

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways, including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host's IFN-I response, West Nile virus (WNV) replication is sensitive to pretreatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV's sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells, WNV replication is significantly increased and IFN-I induction and signaling are impaired compared to wild-type (wt) cells. IFN-ß pretreatment was more effective in protecting against subsequent WNV infection in wt cells than TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next-generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced JAK1 and STAT1 phosphorylation and impaired induction of several interferon-stimulated genes (ISGs) following WNV infection or IFN-ß treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function are dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identify VAMP8 as a novel regulator of the IFN-I system.IMPORTANCE WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.

Hydrogen cyanide produced by the soil bacterium Chromobacterium sp. Panama contributes to mortality in Anopheles gambiae mosquito larvae.

Mosquito larvae continuously encounter microbes in their aquatic environment, which serve as food and play a critical role in successful development. In previous work, we isolated a Chromobacterium sp. (C.sp_P) with larvicidal activity from the midgut of dengue vector Aedes mosquitoes in Panama. In this study, we found a positive correlation between initial concentrations of C.sp_P and larval mortality rates, and that C.sp_P is more efficient at inducing larval mortality in a high nutrient environment. Multiple Chromobacterium species induce larval mortality with similar efficacy to C.sp_P except for C. subtsugae. We also found that a non-lethal dose of C.sp_P lengthens development time and increases mortality over multiple developmental stages, suggesting persistent effects of exposure. Additionally, we showed that larvicidal activity persists in the larval breeding water after removal of live bacteria, and that the larvicidal factor in C.sp_P-treated water is smaller than 3 kDa, heat resistant to 90 °C, and lost after vacuum centrifugation. We showed that C.sp_P produces hydrogen cyanide in culture and in larval water at concentrations sufficient to kill An. gambiae larvae, and treatment of the larval water with a cyanide antidote eliminated larvicidal activity. We conclude that a potential mechanism by which C.sp_P can induce larval mortality is via production of hydrogen cyanide.

The mosquito adulticidal Chromobacterium sp. Panama causes transgenerational impacts on fitness parameters and elicits xenobiotic gene responses.

BACKGROUND: Vector control is critical in reducing the disease burden caused by mosquitoes, and insecticides are an effective tool to control vector populations. Resistance to common insecticides is now widespread, and novel classes of insecticides are needed. In previous work, we described the mosquitocidal activity of Chromobacterium sp. Panama (C.sp_P), a bacterium found in association with mosquitoes in natural populations. In the current work, we further explored the effects of exposure to the bacterium on mosquito fitness and mosquito physiology. RESULTS: We found that C.sp_P has mosquitocidal activity against a broad range of mosquito taxa. When exposed to C.sp_P as adults, female An. gambiae suffered reduced longevity, but experienced no change in fecundity. The offspring of these females, however, had higher mortality as larvae and were slower to develop compared to offspring of control females. We also found that the mosquitocidal activity of C.sp_P was retained after removal of live cells from biofilm culture media, suggesting the bacteria secrete mosquitocidal compound(s) into the media during growth. Exposure to this cell-free C.sp_P-conditioned media caused female midgut transcriptional changes comprising detoxification, xenobiotic response, and stress response genes, suggesting the physiological response to C.sp_P is similar to that of insecticide exposure. Finally, we found that multiple members of the Chromobacterium genus had mosquitocidal activity, but this activity was highest in mosquitoes treated with C.sp_P. CONCLUSIONS: Our findings suggest that C.sp_P produces factor(s) with strong effects on mosquito longevity and fitness, which may be of interest for mosquitocide development. More generally, they indicate that further exploration of mosquito-associated and environmental microbes for novel insecticidal compounds or biocontrol agents is warranted.

The TRIMendous Role of TRIMs in Virus-Host Interactions.

The innate antiviral response is integral in protecting the host against virus infection. Many proteins regulate these signaling pathways including ubiquitin enzymes. The ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes work together to link ubiquitin, a small protein, onto other ubiquitin molecules or target proteins to mediate various effector functions. The tripartite motif (TRIM) protein family is a group of E3 ligases implicated in the regulation of a variety of cellular functions including cell cycle progression, autophagy, and innate immunity. Many antiviral signaling pathways, including type-I interferon and NF-κB, are TRIM-regulated, thus influencing the course of infection. Additionally, several TRIMs directly restrict viral replication either through proteasome-mediated degradation of viral proteins or by interfering with different steps of the viral replication cycle. In addition, new studies suggest that TRIMs can exert their effector functions via the synthesis of unconventional polyubiquitin chains, including unanchored (non-covalently attached) polyubiquitin chains. TRIM-conferred viral inhibition has selected for viruses that encode direct and indirect TRIM antagonists. Furthermore, new evidence suggests that the same antagonists encoded by viruses may hijack TRIM proteins to directly promote virus replication. Here, we describe numerous virus-TRIM interactions and novel roles of TRIMs during virus infections.

Antigenic and Biological Characterization of ORF2-6 Variants at Early Times Following PRRSV Infection.

Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) challenges efforts to develop effective and broadly acting vaccines. Although genetic variation in PRRSV has been extensively documented, the effects of this variation on virus phenotype are less well understood. In the present study, PRRSV open reading frame (ORF)2-6 variants predominant during the first six weeks following experimental infection were characterized for antigenic and replication phenotype. There was limited genetic variation during these early times after infection; however, distinct ORF2-6 haplotypes that differed from the NVSL97-7895 inoculum were identified in each of the five pigs examined. Chimeric viruses containing all or part of predominant ORF2-6 haplotypes were constructed and tested in virus neutralization and in vitro replication assays. In two pigs, genetic variation in ORF2-6 resulted in increased resistance to neutralization by autologous sera. Mapping studies indicated that variation in either ORF2-4 or ORF5-6 could confer increased neutralization resistance, but there was no single amino acid substitution that was predictive of neutralization phenotype. Detailed analyses of the early steps in PRRSV replication in the presence and absence of neutralizing antibody revealed both significant inhibition of virion attachment and, independently, a significant delay in the appearance of newly synthesized viral RNA. In all pigs, genetic variation in ORF2-6 also resulted in significant reduction in infectivity on MARC-145 cells, suggesting variation in ORF2-6 may also be important for virus replication in vivo. Together, these data reveal that variation appearing early after infection, though limited, alters important virus phenotypes and contributes to antigenic and biologic diversity of PRRSV.