An interaction between the orthopoxvirus glycoproteins A34 and B5 has been reported. The transmembrane and ectodomain of A34 are sufficient for interaction with B5, localization of B5 to the site of intracellular wrapping, and subsequent incorporation into the envelope of released extracellular virions. Several mutagenic approaches were undertaken to better define the B5 interaction domain on A34. A set of C-terminal truncations in A34 identified residues 1 to 80 as sufficient for interaction with B5. Additional truncations identified residues 80 to 130 of A34 as sufficient for interaction with B5. To better understand the function of this region, a set of recombinant viruses expressing A34 with the full, partial, or no B5 interaction site (residues 1 to 130, 1 to 100, and 1 to 70, respectively) was constructed. All the recombinants expressing truncations of A34 incorporated B5 into extracellular virions but had a small-plaque phenotype similar to that of a virus with the A34R gene deleted (vΔA34R). Further characterization indicated that the small-plaque phenotype exhibited by these viruses is due to a combination of abrogated actin tail formation, reduced cell binding, and a defect in polyanion-induced nonfusogenic dissolution. Taken together, these results suggest that residues 80 to 130 of A34 are not necessary for the proper localization and incorporation of B5 into extracellular virions and, furthermore, that the C-terminal residues of A34 are involved in cell binding and dissolution. IMPORTANCE Previous studies have shown that the vaccinia virus glycoproteins A34 and B5 interact, and in the absence of A34, B5 is mislocalized and not incorporated into extracellular virions. Here, using a transient-transfection assay, residues 80 to 130 of the ectodomain of A34 were determined to be sufficient for interaction with B5. Recombinant viruses expressing A34 with a full, partial, or no B5 interaction site were constructed and characterized. All of the A34 truncations interacted with B5 as predicted by the transient-transfection studies but had a small-plaque phenotype. Further analysis revealed that all of the recombinants incorporated detectable levels of B5 into released virions but were defective in cell binding and extracellular virion (EV) dissolution. This study is the first to directly demonstrate that A34 is involved in cell binding and implicate the ectodomain in this role.

Orthopoxviruses produce two, antigenically distinct, infectious enveloped virions termed intracellular mature virions and extracellular virions (EV). EV have an additional membrane compared to intracellular mature virions due to a wrapping process at the trans-Golgi network, and are required for cell-to-cell spread and pathogenesis. Specific to the EV membrane is a number of proteins highly conserved among orthopoxviruses, including F13, which is required for the efficient wrapping of intracellular mature virions to produce EV, and plays a role in EV entry. The distantly related molluscipoxvirus, molluscum contagiosum virus, is predicted to encode several vaccinia virus homologs of EV specific proteins, including the homolog of F13L, MC021L. To study the function of MC021, we replaced the F13L open reading frame in vaccinia virus with an epitope tagged version of MC021L. The resulting virus (vMC021L-HA) had a small plaque phenotype compared to vF13L-HA, but larger than vΔF13L. Localization of MC021-HA was markedly different from that of F13-HA in infected cells, but MC021-HA was still incorporated in the EV membrane. Similar to F13-HA, MC021-HA was capable of interacting with both A33 and B5. Although MC021-HA expression did not fully restore plaque size, vMC021L-HA produced similar amounts of EV compared to vF13L-HA, suggesting that MC021 retained some of the functionality of F13. Further analysis revealed that EV produced from vMC021L-HA exhibit a marked reduction in target cell binding, and an increase in dissolution, both of which correlated with a small plaque phenotype. IMPORTANCE The vaccinia virus extracellular virion protein F13 is required for the production and release of infectious extracellular virus, which in-turn, is essential for subsequent spread and pathogenesis of orthopoxviruses. Molluscum contagiosum virus infects millions of people worldwide each year, but it is unknown whether EV is produced during infection for spread. Molluscum contagiosum virus contains a homolog of F13L, termed MC021L. To study the potential function of this homolog during infection, we utilized vaccinia virus as a surrogate, and we show that a vaccinia virus expressing MC021L-HA in place of F13L-HA exhibits a small plaque phenotype but produces similar levels of EV. These results suggest that MC021-HA can compensate for the loss of F13-HA by facilitating wrapping to produce EV, and further delineates the dual role of F13 during infection.

Orthopoxviruses produce two, antigenically distinct, infectious enveloped virions termed intracellular mature virions and extracellular virions. Extracellular virions are required for cell-to-cell spread and pathogenesis. Specific to the extracellular virion membrane, glycoproteins A33, A34, and B5 are highly conserved among orthopoxviruses and have roles during extracellular virion formation and subsequent infection. B5 is dependent on an interaction with either A33 or A34 for localization to the site of intracellular envelopment and incorporation into the envelope of released extracellular virions. In this report we show that an interaction between A33 and A34 can be detected in infected cells. Furthermore, we show that a three-protein complex between A33, A34, and B5 forms in the endoplasmic reticulum (ER) that disassociates post ER export. Finally, immunofluorescence reveals that coexpression of all three glycoproteins results in their localization to a juxtanuclear region that is presumably the site of intracellular envelopment. These results demonstrate the existence of two previously unidentified interactions: one between A33 and A34 and another simultaneous interaction between all three of the glycoproteins. Furthermore, these results indicate that interactions among A33, A34, and B5 are vital for proper intracellular trafficking and subcellular localization. IMPORTANCE The secondary intracellular envelopment of poxviruses at the trans-Golgi network to release infectious extracellular virus (EV) is essential for their spread and pathogenesis. Viral glycoproteins A33, A34, and B5 are critical for the efficient production of infectious EV and interactions among these proteins are important for their localization and incorporation into the outer extracellular virion membrane. We have uncovered a novel interaction between glycoproteins A33 and A34. Furthermore, we show that B5 can interact with the A33-A34 complex. Our analysis indicates that the three-protein complex has a role in ER exit and proper localization of the three glycoproteins to the intracellular site of wrapping. These results show that a complex set of interactions occur in the secretory pathway of infected cells to ensure proper glycoprotein trafficking and envelope content, which is important for the release of infectious poxvirus virions.

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.

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