A gammaherpesviral internal repeat contributes to latency amplification.

BACKGROUND: Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression.

Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3.

Different pathways of bilayer disruption by the structurally related antimicrobial peptides cecropin B, B1 and B3, revealed by surface plasma resonance analysis of immobilized liposomes, differential scanning calorimetry of peptide-large unilamellar vesicle interactions, and light microscopic analysis of peptide-treated giant unilamellar vesicles, have been identified in this study. Natural cecropin B (CB) has one amphipathic and one hydrophobic alpha-helix, whereas cecropins B1 (CB1) and B3 (CB3), which are custom-designed, chimaeric analogues of CB, possess either two amphipathic or two hydrophobic alpha-helices, respectively. Surface plasma resonance analysis of unilamellar vesicles immobilized through a biotin-avidin interaction showed that both CB and CB1 bind to the lipid bilayers at high concentration (>10 microm); in contrast, CB3 induces disintegration of the vesicles at all concentrations tested. Differential scanning calorimetry showed the concentration-dependent effect of bilayer disruption, based on the different thermotrophic phase behaviours and the shapes of the thermal phase-transition curves obtained. The kinetics of the lysis of giant unilamellar vesicles observed by microscopy demonstrated that both CB and CB1 effect a continuous process involving loss of integrity followed by coalescence and resolution into smaller vesicles, whereas CB3 induces rapid formation of irregular-shaped, nonlamellar structures which rapidly disintegrate into twisted, microtubule-containing debris before being completely destroyed. On the basis of these observations, models by which CB, CB1 and CB3 induce lysis of lipid bilayers are discussed.