12 research outputs found
Binding of hnRNP I–vRNA Regulates Sindbis Virus Structural Protein Expression to Promote Particle Infectivity
Alphaviruses cause significant outbreaks of febrile illness and debilitating multi-joint arthritis for prolonged periods after initial infection. We have previously reported that several host hnRNP proteins bind to the Sindbis virus (SINV) RNAs, and disrupting the sites of these RNA–protein interactions results in decreased viral titers in tissue culture models of infection. Intriguingly, the primary molecular defect associated with the disruption of the hnRNP interactions is enhanced viral structural protein expression; however, the precise underlying mechanisms spurring the enhanced gene expression remain unknown. Moreover, our previous efforts were unable to functionally dissect whether the observed phenotypes were due to the loss of hnRNP binding or the incorporation of polymorphisms into the primary nucleotide sequence of SINV. To determine if the loss of hnRNP binding was the primary cause of attenuation or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to restore the binding of the hnRNP proteins in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction by incorporating the 20nt bovine immunodeficiency virus transactivation RNA response (BIV-TAR) at the site of the native hnRNP I interaction sequence, which will bind with high specificity to proteins tagged with a TAT peptide. The reestablishment of the hnRNP I–vRNA interaction via the BIV-TAR/TAT tethering approach restored the phenotype back to wild-type levels. This included an apparent decrease in structural protein expression in the absence of the native primary nucleotide sequences corresponding to the hnRNP I interaction site. Collectively, the characterization of the hnRNP I interaction site elucidated the role of hnRNPs during viral infection
An Assembly-Activating Site in the Hepatitis B Virus Capsid Protein Can Also Trigger Disassembly
The Hepatitis B Virus (HBV) core
protein homodimers self-assemble to form an icosahedral capsid that
packages the viral genome. Disassembly occurs in the nuclear basket
to release the mature genome to the nucleus. Small molecules have
been developed that bind to a pocket at the interdimer interface to
accelerate assembly and strengthen interactions between subunits;
these are under development as antiviral agents. Here, we explore
the role of the dimer–dimer interface by mutating sites in
the drug-binding pocket to cysteine and examining the effect of covalently linking
small molecules to them. We find that ligands bound to the pocket
may trigger capsid disassembly in a dose-dependent manner. This result
indicates that, at least transiently, the pocket adopts a destabilizing
conformation. We speculate that this pocket also plays a role in virus
disassembly and genome release by binding ligands that are incompatible
with virus stability, “unwanted guests.” Investigating
protein–protein interactions, especially large protein polymers,
offers new and unique challenges. By using an engineered addressable
thiol, we provide a means to examine the effects of modifying an interface
without requiring drug-like properties for the ligand
Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System
<u>C</u>lustered <u>R</u>egularly <u>I</u>nterspaced <u>S</u>hort <u>P</u>alindromic <u>R</u>epeats (CRISPR) RNA-guided endonucleases
are powerful new tools for targeted genome engineering. These nucleases
provide an efficient and precise method for manipulating eukaryotic
genomes; however, delivery of these reagents to specific cell-types
remains challenging. Virus-like particles (VLPs) derived from bacteriophage
P22, are robust supramolecular protein cage structures with demonstrated
utility for cell type-specific delivery of encapsulated cargos. Here,
we genetically fuse Cas9 to a truncated form of the P22 scaffold protein,
which acts as a template for capsid assembly as well as a specific
encapsulation signal for Cas9. Our results indicate that Cas9 and
a single-guide RNA are packaged inside the P22 VLP, and activity assays
indicate that this RNA-guided endonuclease is functional for sequence-specific
cleavage of dsDNA targets. This work demonstrates the potential for
developing P22 as a delivery vehicle for cell specific targeting of
Cas9