Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant.

Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.

Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities.

Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.

Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4.

Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid.

Vaccinia virions deficient in transcription enzymes lack a nucleocapsid.

The poxvirus virion contains an inner tubular nucleocapsid structure. The nucleocapsid is apparently labile to conventional electron microscopy fixation procedures and has therefore been largely ignored for decades. Advancements in electron microscopy sample preparation, notably high pressure freezing, better preserve the nucleocapsid structure. Using high pressure freezing and electron microscopy, we have compared the virion structures of wt virus and mutant viruses known to be deficient in packaging of viral transcription enzymes. We show that the mutant viruses lack a defined nucleocapsid. These results support the hypothesis that the nucleocapsid contains the viral DNA genome complexed with viral transcription enzymes and structural proteins. The studies open the door to further investigation of the composition and ultrastructure of the poxvirus nucleocapsid.