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Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

Current efforts to develop an Epstein-Barr virus subunit vaccine are based on the major envelope glycoprotein gp340. Given the central role of CD4+ T cells in regulating immune responses to subunit vaccine antigens, the present study has begun the work of identifying linear epitopes which are recognized by human CD4+ T cells within the 907-amino-acid sequence of gp340. A panel of gp340-specific CD4+ T-cell clones from an Epstein-Barr virus-immune donor were first assayed for their proliferative responses to a series of truncated gp340 molecules expressed from recombinant DNA vectors in rat GH3 cells, by using an autologous B lymphoblastoid cell line as a source of antigen-presenting cells. The first four T-cell clones analyzed all responded to a truncated form of gp340 which contained only the first 260 N-terminal amino acids. These clones were subsequently screened for responses to each of a panel of overlapping synthetic peptides (15-mers) corresponding to the primary amino acid sequence of the first 260 N-terminal amino acids of gp340. One clone (CG2.7) responded specifically to peptides from the region spanning amino acids 61 to 81, while three other clones (CG5.15, CG5.24, and CG5.36) responded specifically to peptides from the region spanning amino acids 163 to 183. Work with individual peptides from these regions allowed finer mapping of the T-cell epitopes and also revealed the highly dose-dependent nature of peptide-induced responses, with inhibitory effects apparent when the most antigenic peptides were present at supraoptimal concentrations. Experiments using homozygous typing B lymphoblastoid cell lines as antigen-presenting cells showed that the T-cell clones with different epitope specificities were restricted through different HLA class II antigens; clone CG2.7 recognized epitope 61-81 in the context of HLA DRw15, whereas clones CG5.15, CG5.24, and CG5.36 recognized epitope 163-183 in the context of HLA DRw11. The present protocol therefore makes a systematic analysis of CD4+ T-cell epitopes within gp340 possible; it will be necessary to screen gp340-specific T-cell clones from a variety of donors to assess the wider influence of HLA class II polymorphism upon epitope choice.

Gel analysis of vaccinia virus particles purified by buoyant [correction of bouyant] density demonstrates a protein with an estimated molecular mass of 59 kDa, which is apparently restricted to the intracellular mature virion (IMV) form. Western blotting (immunoblotting) and immunoprecipitation procedures identify the protein as the vaccinia virus 4c protein, which facilitates occlusion of poxvirus particles within cowpox cytoplasmic inclusions. Western blotting procedures also identify the truncated A-type inclusion protein of vaccinia virus as a specific marker for IMV particles. Kinetic analyses of virion maturation and 4c production suggest that peak enveloped virion production occurs before peak IMV production in the virus replication cycle and that 4c production is concomitant with maturation of IMV. The implications for a distinct and evolutionarily conserved function of IMV in viral pathogenesis are discussed.