Nucleation seed size determines amyloid clearance and establishes a barrier to prion appearance in yeast.

Amyloid appearance is a rare event that is promoted in the presence of other aggregated proteins. These aggregates were thought to act by templating the formation of an assembly-competent nucleation seed, but we find an unanticipated role for them in enhancing the persistence of amyloid after it arises. Specifically, Saccharomyces cerevisiae Rnq1 amyloid reduces chaperone-mediated disassembly of Sup35 amyloid, promoting its persistence in yeast. Mathematical modeling and corresponding in vivo experiments link amyloid persistence to the conformationally defined size of the Sup35 nucleation seed and suggest that amyloid is actively cleared by disassembly below this threshold to suppress appearance of the [PSI+] prion in vivo. Remarkably, this framework resolves multiple known inconsistencies in the appearance and curing of yeast prions. Thus, our observations establish the size of the nucleation seed as a previously unappreciated characteristic of prion variants that is key to understanding transitions between prion states.

Inhibition of endocytic pathways impacts cytomegalovirus maturation.

Endocytic processes are critical for cellular entry of several viruses; however, the role of endocytosis in cellular trafficking of viruses beyond virus entry is only partially understood. Here, we utilized two laboratory strains (AD169 and Towne) of human cytomegalovirus (HCMV), which are known to use cell membrane fusion rather than endocytosis to enter fibroblasts, in order to study a post-entry role of endocytosis in HCMV life cycle. Upon pharmacological inhibition of dynamin-2 or clathrin terminal domain (TD) ligand association, these strains entered the cells successfully based on the expression of immediate early viral protein. However, both the inhibitors significantly reduced the growth rates and final virus yields of viruses without inhibiting the expression of early to late viral proteins. Clathrin accumulated in the cytoplasmic virus assembly compartment (vAC) of infected cells co-localizing with virus tegument protein pp150 and the formation of vAC was compromised upon endocytic inhibition. Transmission electron micrographs (TEM) of infected cells treated with endocytosis inhibitors showed intact nuclear stages of nucleocapsid assembly but the cytoplasmic virus maturation was greatly compromised. Thus, the data presented here implicate endocytic pathways in HCMV maturation and egress.

Highly acidic C-terminal region of cytomegalovirus pUL96 determines its functions during virus maturation independently of a direct pp150 interaction.

UNLABELLED: Tegument proteins pp150 and pUL96 function at a late step in cytomegalovirus (CMV) maturation. Here, we show that pp150 interacts directly with pUL96; however, the N-terminal region of pp150 and the C-terminal region of pUL96, which are critical for these proteins to function, are not required for this interaction. Moreover, the largely dispensable C-terminal region of pp150 is critical for pp150-pUL96 interaction. To further study the role of pUL96, several point and clustered mutations were engineered into the CMV Towne bacterial artificial chromosome (Towne-BAC) genome, replacing the conserved negatively charged C-terminal residues of pUL96. Although individual point mutations (E122A, D124A, and D125A) reduced virus growth slightly, the clustered mutations of 122EVDDAV127 significantly reduced virus growth, produced small syncytial plaque phenotypes, and impacted a late stage of virus maturation. When the UL96 C-terminal alanine conversion mutant (B6-BAC) virus was serially passaged in cell culture, it gained a plaque size comparable to that of Towne-BAC, displayed an altered restriction fragment length pattern, and replicated with increased growth kinetics. Whole-genome sequencing of this passaged virus (UL96P10) and the similarly passaged Towne-BAC virus revealed major differences only in the RNA4.9 and UL96 regions. When one of the mutations in the UL96 coding region was engineered into the B6-BAC virus, it significantly increased the plaque size and rescued the virus growth rate. Thus, accumulation of compensatory mutations only in UL96 in this revertant and the specific involvement of functionally dispensable regions of pp150 in the pUL96-pp150 interaction point toward a role for pUL96 in virus maturation that does not depend upon pp150. IMPORTANCE: Human cytomegalovirus causes significant medical problems in newborns, as well as in people with low immunity. In this study, we investigated the functions of two essential virus proteins, pp150 and pUL96, and determined the impact of their mutual interaction on virus replication. These studies provide valuable information that is critical for the development of targeted antiviral therapies.

Complete Genome Sequence of a UL96 Mutant Cytomegalovirus Towne-BAC (Bacterial Artificial Chromosome) Isolate Passaged in Fibroblasts To Allow Accumulation of Compensatory Mutations.

Here, we present the complete genome sequence of a cytomegalovirus Towne-BAC (bacterial artificial chromosome) isolate that we first genetically engineered to mutate the UL96 gene and then serially passaged in human fibroblasts to allow for the accumulation of compensatory mutations. A total of 17 single-base substitutions were discovered in the passaged genome compared to the reference sequence (KF493877).