The Kielian Lab - Research

VIRUS ENTRY AND EXIT

Our research focuses on the molecular mechanisms of virus entry and exit. Our experimental systems include alphaviruses, rubella virus, and flaviviruses. These viruses are small enveloped viruses that contain plus-sense RNA genomes and include important human pathogens such as Chikungunya, rubella, and dengue viruses. We use a wide variety of experimental approaches including molecular biology, virology, cell biology, live cell imaging, protein biochemistry, mapping of RNA binding sites, and structural analysis.

Alphavirus lifecycle.

Alphaviruses, rubella virus and flaviviruses all enter cells by endocytic uptake and then fuse their membrane with the endosome membrane in a reaction triggered by the low pH of the endocytic vesicle. RNA replication occurs in the cytoplasm and new progeny viruses bud at the plasma membrane (as shown for alphaviruses), or into the Golgi compartment or the endoplasmic reticulum (for rubella and flaviviruses, respectively).

How are virus fusion proteins regulated during entry and exit?

During alphavirus and flavivirus biogenesis, a companion protein forms a closely-associated dimer with the fusion protein, and protects it from low pH and premature fusion during exocytic transport. This companion protein must then dissociate to permit virus fusion. The pH protection mechanisms for many other viruses are unknown, and we are using Rubella virus as a system to define novel mechanisms of pH protection. We are also investigating the pH-dependent control mechanisms for the Rubella virus fusion reaction.

How do viruses assemble and bud?

Alphaviruses bud from the plasma membrane to produce highly organized particles containing 1 genomic RNA molecule and 240 copies each of the (internal) capsid protein and the E1 and E2 membrane proteins. Little is known about alphavirus assembly and budding, although it is clear that these processes are highly regulated to produce organized virus particles of high specific infectivity.

How does the alphavirus capsid protein specifically package and assemble with the viral RNA?

Alphaviruses RNA-capsid binding studies have defined interaction sites that are enriched on the genomic RNA, leading to its preferential packaging and assembly into nucleocapsid cores. The data suggest that alphavirus RNA packaging uses multiple interactions to promote efficient packaging.

Key questions in virus assembly and budding:

• Structure of capsid protein + specific RNA?

• Where/how do envelope proteins and capsid interact?

• Transport mechanism for capsid/nucleocapsid?

• Regulation of nucleocapsid assembly?

• Role of host cell proteins in nucleocapsid assembly, transport, and budding?

• Inhibitors of nucleocapsid assembly, budding?

An alternative pathway to “free” alphavirus budding and exit: Long intercellular extensions are induced by alphavirus infection.

We have developed fluorescently tagged alphaviruses to follow virus assembly and budding in real time in infected cells. These studies revealed that alphaviruses can spread from cell to cell via long intercellular extensions that form stable contacts between an infected cell and neighboring cells. Formation of extensions can alao be induced by expression of the alphavirus structural proteins—virus infection is not required. The interaction of E2 and the capsid protein is required, and virus transmission requires the budding of fusion-active viruses.

Key questions for intercellular extensions:

• E2 domain involved?

• Role of capsid-E2 interaction?

• Ligand(s) on the target cell, adhesion molecules?

• Selective budding at the extension tip?

• Signaling pathways, timing, mechanism of actin and microtubule reorganization?

• Importance in vivo (antibody protection, virulence)?

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