Bertram L. Jacobs

Orthopoxvirus monkeypox (MPXV) is a zoonotic pathogen with two major clades that cause outbreaks in humans. Differentiation between the historically more clinically severe clade I and clade IIb is needed for effective public health response, such as in the ongoing 2022-2025 MPXV multi-country outbreaks. We have developed three loop-mediated isothermal amplification (LAMP) assays that differentiate between clade I, clade IIa/IIb MPXV, and broader Orthopoxvirus species by exploiting viral genomic rearrangements and amplicon length limitations inherent to LAMP. We demonstrate clade specificity between clade I and clade II viral cell culture lysates and rapid detection of crude lesion specimens isolated from 45 patients during the latest MPXV USA 2022-2025 outbreaks. MPXV genomic DNA can be detected in as little as 10 minutes and clade-specific results detected in under an hour with limited instrumentation. We adapt a portable microfluidic system for point-of-need outbreak responses to zoonotic orthopoxvirus.

Nonsense-mediated decay (NMD) is an mRNA decay pathway which degrades potential harmful transcripts that contain premature termination codons. However, NMD’s importance also extends to the control of isoform abundance under physiological conditions. During viral infection, NMD is inhibited through numerous mechanisms; however, NMD has been shown to have both antiviral as well as proviral activities, raising further questions into the role and control of NMD during viral infection. These observations have led us to investigate the potential involvement of NMD in dsRNA sensing as a mechanism that might explain these discrepancies. Using EIF4A2 exon 10B inclusion as an example of AS-NMD isoform accumulating during viral infection, we show that dsRNA sensing inhibits NMD. This effect is correlated with translational blockade and is driven primarily by RNaseL activation, and by PKR in the absence of RNaseL activation. Surprisingly, NMD inhibition limits the induction of IFN-β as well as interferon-stimulated genes, and this effect is upstream of IRF3 phosphorylation and translocation to the nucleus. NMD inhibition also decreases PKR and RNaseL activation as well as PIC-mediated cell death by decreasing the dsRNA content, suggesting NMD directly controls dsRNA sensing by controlling the dsRNA load. Therefore, inhibition of NMD upon dsRNA sensing provides a negative feedback loop that contributes to shaping the innate immune sensing pathways.

The nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting the XPO1-mediated nuclear export pathway with selective inhibitors has a diverse effect on virus replication by regulating antiviral, proviral, and anti-inflammatory pathways. The XPO1 inhibitor Selinexor is an FDA-approved anticancer drug predicted to have antiviral or proviral functions against viruses. Here, we observed that the pretreatment of cultured cell lines from human or mouse origin with the nuclear export inhibitor Selinexor significantly enhanced the protein expression and replication of mouse hepatitis virus (MHV), a mouse coronavirus. The knockdown of cellular XPO1 protein expression also significantly enhanced the replication of MHV in human cells. However, for SARS-CoV-2, Selinexor treatment had diverse effects on virus replication in different cell lines. These results indicate that XPO1-mediated nuclear export pathway inhibition might affect coronavirus replication depending on cell types and virus origin.

Monkeypox/mpox virus (MPXV) has re-emerged as the most important orthopoxvirus infection of humans. Despite being pathogenic, it contains a natural truncation at the N-terminus of its homologue of the vaccinia virus (VACV) innate immune evasion protein, E3, leading to the loss of the first 37 amino acids. Our previous data have shown that VACV E3 protein is required for interferon-resistance of VACV. The N-terminal Z-nucleic acid binding domain is necessary to inhibit induction of necroptosis, as VACV containing an N-terminal deletion (VACV-E3LΔ37N) undergoes rapid ZBP1-dependent necroptotic cell death, through activation of RIPK3, and subsequent phosphorylation and trimerization of the executioner of necroptosis, MLKL. Despite lacking parts of the N-terminus, MPXV has evolved ways in circumventing necroptotic cell death in mouse L929 cells. Our data shows that MPXV infection inhibits phosphorylation of MLKL and does not lead to MLKL trimerization, nor cell death. We show that MPXV inhibits necroptosis in two steps: by degrading RIPK3 through either a caspase-dependent pathway or through the “viral inducer of RIPK3 degradation” (vIRD) and by inhibiting MLKL aggregation. Additionally, MPXV shows interferon sensitivity that is fully ZBP1- and RIPK3- and MLKL-dependent, but independent of necroptosis. Addition of a pancaspase inhibitor (zVAD), or a proteasome inhibitor (MLN4924) does not increase cell death but leads to an increase in interferon sensitivity comparable to VACV-E3LΔ37N. Thus, treatment with an interferon inducer along with a pancaspase or proteasome inhibitor could potentially be a beneficial treatment against MPXV infections.

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille’s Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

Cellular stress granules arise in cells subjected to stress and promote cell survival. A cellular protein that localizes to stress granules is Z-DNA–binding protein 1 (ZBP1), which plays a major role in necroptosis, a programmed cell death pathway mediated by the kinase RIPK3. Here, we showed that the stress granule inducer arsenite activated RIPK3-dependent necroptosis. This pathway required ZBP1, which localized to arsenite-induced stress granules. RIPK3 localized to stress granules in the presence of ZBP1, leading to the formation of ZBP1-RIPK3 necrosomes, phosphorylation of the RIPK3 effector MLKL, and execution of necroptosis. Cells that did not form stress granules did not induce necroptosis in response to arsenite. Together, these results show that arsenite induces ZBP1-mediated necroptosis in a manner dependent on stress granule formation.

The Omicron SARS-CoV-2 variant has been designated as a variant of concern because its spike protein is heavily mutated. In particular, the Omicron spike is mutated at five positions (K417, N440, E484, Q493, and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501Y_MA30, contains a spike that is also heavily mutated, with mutations at four of the five positions in the Omicron spike associated with neutralizing antibody escape (K417, E484, Q493, and N501). In this manuscript, we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against symptoms and death from SARS2-N501Y_MA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that the Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus—administered without parenteral injection—can fully protect against the heavily mutated mouse-adapted SARS2-N501Y_MA30.

Poxviridae have developed a plethora of strategies to evade innate and adaptive immunity. In this review, we focused on the vaccinia virus E3 protein, encoded by the E3L gene. E3 is present within the Chordopoxvirinae subfamily (with the exception of the avipoxviruses and molluscum contagiosum virus) and displays pleiotropic effects on the innate immune system. Initial studies identified E3 as a double-stranded RNA (dsRNA)-binding protein (through its C terminus), able to inhibit the activation of protein kinase dependent on RNA (PKR) and the 2′5′-oligoadenylate synthetase (OAS)/RNase L pathway, rendering E3 a protein counteracting the type I interferon (IFN) system. In recent years, N-terminal mutants of E3 unable to bind to Z-form nucleic acids have been shown to induce the cellular death pathway necroptosis. This pathway was dependent on host IFN-inducible Z-DNA-binding protein 1 (ZBP1); full-length E3 is able to inhibit ZBP1-mediated necroptosis. Binding to what was identified as Z-RNA has emerged as a novel mechanism of counteracting the type I IFN system and has broadened our understanding of innate immunity against viral infections. This article gives an overview of the studies leading to our understanding of the vaccinia virus E3 protein function and its involvement in viral pathogenesis. Furthermore, a short summary of other viral systems is provided.

The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501Y _MA30 , contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501). In this manuscript we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against disease and death from SARS2-N501Y _MA30 . Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus, administered without parenteral injection can fully protect against the heavily mutated mouse-adapted SARS2-N501Y _MA30 .