Abstract
Vaccinia virus (VACV) produces two morphologically distinct mature virions: enveloped virus (EV) and intracellular mature virus (IMV). EV is produced first in the replication cycle and is required for virus dissemination. IMV is thought to be produced by default, when the cellular membranes that wrap EV become exhausted and as the more robust particle, is involved in host-to-host transmission. The aim of this study was to examine the hypothesis that IMV production is not a default, but is an active process mediated by the IMV-specific protein, A26. Two recombinant VACVs were constructed: vldA26L and vldA26L. neo, in which the A26L gene was deleted or inactivated. One-step growth curves demonstrated a reduction in virus yields for these viruses. Examination of the kinetics of EV and IMV production by the A26L deletion mutants did not demonstrate a reduction in IMV as expected, yet the production of single-membraned virus was perturbed when compared to wild-type VACV IHD-J and a revertant virus. Constitutive expression of A26 from a synthetic early/late promoter did not affect virus yields and did not result in increased production of IMV in favour of EV. Treatment of virions with membrane perturbing agents demonstrated increased susceptibility of EV to inactivation when compared to IMV, suggesting that the EV pre-cursor and mature IMV are different particles. Mutant IMV from A26L-VACV cultures demonstrated increased sensitivity to the detergent Tween-20 when compared to wild-type IMV, behaving similarly to stripped EV. This suggests resistance might be attributable to the presence of IMV-specific proteins that are not found on stripped EV. This study provides evidence that IMV is a true end-stage product of VACV morphogenesis, and that OPVs employ a mechanism to actively differentiate particles towards IMV, thus preventing maturation of a subset of virions as EV. This results in two non-interchangeable isoforms of mature infectious virions that are produced to perform their individual functions in the virus life-cycle.
Published Version
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