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The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L, results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity., Competing Interests: The authors have declared that no competing interests exist.

Little is known about whether type 1 (IFNγ), 2 (IL-4/IL-13), or 3 (IL-17A/IL-22) cytokines affect the susceptibility of keratinocytes (KC) to viruses. These immune pathways predominate in various skin diseases: lupus, atopic dermatitis (AD), and psoriasis, respectively. Janus kinase inhibitors (JAKi) are approved to treat both AD and psoriasis, and are in clinical development for lupus. We evaluated whether these cytokines alter viral susceptibility of KC and determined if this effect is modulated by treatment with JAKi. Viral susceptibility to vaccinia virus (VV) or herpes simplex virus-1 (HSV-1) ± JAKi was assessed in immortalized and primary human KC pretreated with cytokines. Exposure to type 2 (IL-4 + IL-13) or the type 3 (IL-22) cytokines significantly increased KC viral susceptibility. Specifically, there was a peak increase of 12.2 ± 3.1-fold (IL-4 + IL-13) or 7.7 ± 2.8-fold (IL-22) in VV infection as measured by plaque number. Conversely, IFNγ significantly reduced susceptibility to VV (63.1 ± 64.4-fold). The IL-4 + IL-13-induced viral susceptibility was reduced (44 ± 16%) by JAK1 inhibition, while the IL-22-enhanced viral susceptibility was diminished (76 ± 19%) by TYK2 inhibition. IFNγ-mediated resistance to viral infection was reversed by JAK2 inhibition (366 ± 294% increase in infection). Cytokines expressed in AD skin (IL-4, IL-13, IL-22) increase KC viral susceptibility while IFNγ is protective. JAKi that target JAK1 or TYK2 reversed cytokine-enhanced viral susceptibility, while JAK2 inhibition reduced the protective effects of IFNγ. [ABSTRACT FROM AUTHOR]

Individuals with underlying chronic skin conditions, notably atopic dermatitis (AD), are disproportionately affected by infections from members of the herpesviridae, papovaviridae, and poxviridae families. Many patients with AD experience recurrent, widespread cutaneous viral infections that can lead to viremia, serious organ complications, and even death. Little is known about how the type 2 inflammatory environment observed in the skin of AD patients impacts the susceptibility of epidermal cells (keratinocytes) to viral pathogens. Herein, we studied the susceptibility of keratinocytes to the prototypical poxvirus, vaccinia virus (VV)—the causative agent of eczema vaccinatum—under conditions that simulate the epidermal environment observed in AD. Treatment of keratinocytes with type 2 cytokines (IL-4 and -13) to simulate the inflammatory environment or a tight junction disrupting peptide to mirror the barrier disruption observed in AD patients, resulted in a differentiation-dependent increase in susceptibility to VV. Furthermore, pan JAK inhibition was able to diminish the VV susceptibility occurring in keratinocytes exposed to type 2 cytokines. We propose that in AD, the increased viral susceptibility of keratinocytes leads to enhanced virus production in the skin, which contributes to the rampant dissemination and pathology seen within patients. [ABSTRACT FROM AUTHOR]

The vaccinia virus protein F13, encoded by the F13L gene, is conserved across the subfamily Chordopoxvirinae and is critical among orthopoxviruses to produce the wrapped form of virus that is required for cell-to-cell spread. F13 is the major envelope protein on the membrane of extracellular forms of virus; however, it is not known if F13 is required in steps postwrapping. In this report, we utilize two temperature-sensitive vaccinia virus mutants from the Condit collection of temperature-sensitive viruses whose small plaque phenotypes have been mapped to the F13L gene. Despite the drastic reduction in plaque size, the temperature-sensitive viruses were found to produce levels of extracellular virions similar to those of the parental strain, Western Reserve (WR), at the permissive and nonpermissive temperatures, suggesting that they are not defective in extracellular virion formation. Analyses of extracellular virions produced by one temperature-sensitive mutant found that those produced at the nonpermissive temperature had undetectable levels of F13 and bound cells with efficiency similar to that of WR but displayed delayed cell entry kinetics. Additionally, low-pH treatment of cells bound by extracellular virions produced at the nonpermissive temperature by the temperature-sensitive reporter virus was unable to overcome a block in infection by bafilomycin A1, suggesting that these virions display increased resistance to dissolution of the extracellular virion envelope. Taken together, our results suggest that F13 plays a role both in the formation of extracellular virions and in the promotion of their rapid entry into cells by enhancing the sensitivity of the membrane to acid-induced dissolution. IMPORTANCE Vaccinia virus (VACV) is an orthopoxvirus and produces two infectious forms, mature virions (MV) and extracellular virions (EV). EV are derived from MV and contain an additional membrane that must first be removed prior to cell entry. F13 is critical for the formation of EV, but a postenvelopment role has not been described. Here, two temperature-sensitive VACV mutants whose deficiencies were previously mapped to the F13L locus are characterized. Both viruses produced EV at the nonpermissive temperature at levels similar to those of a virus that has F13L, yet they had a small plaque phenotype and rate of spread similar to that of an F13L deletion virus. F13 was undetectable on the EV membrane at the nonpermissive temperature, and these EV exhibited delayed cell entry kinetics compared to EV containing F13. This study is the first to conclusively demonstrate a novel role for F13 in cell entry of the EV form of the virus. [ABSTRACT FROM AUTHOR]