A split ribozyme that links detection of a native RNA to orthogonal protein outputs.

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme.

Replication of HIV-1 envelope protein cytoplasmic domain variants in permissive and restrictive cells.

Wild type (WT) HIV-1 envelope (Env) protein cytoplasmic tails (CTs) appear to be composed of membrane-proximal, N-terminal unstructured regions, and three C-terminal amphipathic helices. Previous studies have shown that WT and CT-deleted (ΔCT) Env proteins are incorporated into virus particles via different mechanisms. WT Env proteins traffic to cell plasma membranes (PMs), are rapidly internalized, recycle to PMs, and are incorporated into virions in permissive and restrictive cells in a Gag matrix (MA) protein-dependent fashion.

Analysis of HIV-1 Matrix-Envelope Cytoplasmic Tail Interactions.

The matrix (MA) domains of HIV-1 precursor Gag (PrGag) proteins direct PrGag proteins to plasma membrane (PM) assembly sites where envelope (Env) protein trimers are incorporated into virus particles. MA targeting to PM sites is facilitated by its binding to phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], and MA binding to cellular RNAs appears to serve a chaperone function that prevents MA from associating with intracellular membranes prior to arrival at the PI(4,5)P2-rich PM. Investigations have shown genetic evidence of an interaction between MA and the cytoplasmic tails (CTs) of Env trimers that contributes to Env incorporation into virions, but demonstrations of direct MA-CT interactions have proven more difficult.

Organization of Farnesylated, Carboxymethylated KRAS4B on Membranes.

Mutations of the Ras proteins HRAS, KRAS4A, KRAS4B, and NRAS are associated with a high percentage of all human cancers. The proteins are composed of highly homologous N-terminal catalytic or globular domains, plus C-terminal hypervariable regions (HVRs). Post-translational modifications of all RAS HVRs helps target RAS proteins to cellular membrane locations where they perform their signaling functions.

Lipid biosensor interactions with wild type and matrix deletion HIV-1 Gag proteins.

The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes.