Novel insights into the alphavirus assembly pathway

Jonathan E Snyder, Purdue University

Abstract

Alphaviruses are small, spherical, enveloped positive-sense single stranded RNA viruses responsible for considerable human and animal disease. Alphaviruses form particles of 65-70 nm in diameter, and the icosahedral structures of many alphaviruses have been defined to very high resolutions by cryo-electron microscopy and crystallographic studies, revealing details of the interactions between the structural proteins required for assembly and budding. Nevertheless, much remains to be elucidated with respect to (nucleocapsid) NC assembly, structural protein interactions and trafficking, and the budding process. This disertation contains three distinct studies which, collectively, provide novel insights in the alphavirus assembly pathway. In the first study, I have developed a system to introduce viral NC into live, mammalian cells, using microinjection as a platform, and incorporate these NC into viable virus-like particles (VLPs). Of particular interest is the fact that we can utilize, for the first time, NC assembled in vitro with non-RNA assembly substrates, and incorporate them into such VLPs. Using this system, further studies will examine the viral and host factors involved in transit of and envelopment of NC within an infected cell. Given previously published data detailing the ability to alter alphavirus tropism, our system may allow exploitation of the alphaviruses as a cell-type specific, targeted delivery tool for drugs and nucleic acids. The second study detailed in this thesis involves classical structure-guided mutagenesis. Specifically, a 7Å cryo-EM based reconstruction of the type member of the alphavirus genus, Sindbis (SINV), was recently generated in our lab. Fitting the previously determined crystal structure of the SINV Cp protein ( Cp) into the density map revealed that residue R395 of the cytoplasmic domain of the E2 envelope protein (cdE2) and K252 of the Cp form a putative salt bridge in the virion. Furthermore, a 5-residue flexible loop containing residues K250 and K252 of the Cp was shifted away from cdE2 in the 7Å reconstruction relative to its expected position based on the Cp crystal structure. Given this data, I made multiple amino acid substitutions of K250 and K252 in the SINV Cp and explored the resulting phenotypes. In the context of full length virus, replacing K250 and K252 with negatively charged residues (either D or E) resulted in a lack of virus release as well the absence of NC formation in infected mammalian cells. Surprisingly, however, expressing the same Cp mutants in the mammalian cell without the envelope proteins (in a replicon-based system), or expressing/purifying the mutants from an E. coli expression system and assembling in vitro, yielded NC assembly in all cases. In addition, second-site mutations within cdE2, in the Cp K250/K252 mutant background, restored NC assembly in full-length virus, but not release of infectious particles. Taken together, our data suggests¸ for the first time, a role for the envelope proteins, namely cdE2, in the assembly of SINV NCs during infection in mammalian cells. Finally, we explored the role of a newly described alphavirus structural protein that may be important during virus assembly. Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: Cp, pE2, 6K, and E1. However, ribosomal frameshifting, occurring during translation of the alphavirus structural polyprotein, has been proposed to yield production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the -1 open reading frame (ORF) (Firth et al., 2008). We identified TF in purified preparations of both SINV and Chikungunya (CHIKV) virus particles using a mass spectrometry-based approach. We constructed a panel of SINV mutants that alters the production, size, or sequence of TF, and demonstrate that TF is not absolutely required in culture, although disrupting TF synthesis leads to a decrease in virus release in mammalian and insect cell cultures. In a mouse neuropathogenesis model, animals infected with the TF mutants were protected from mortality unlike the wild type. Using a variety of additional assays, we demonstrated that TF retains ion-channel activity a la 6K, and lack of production of TF does not affect genome synthesis, particle infectivity, or transit of envelope proteins to the cell surface. However, EM analyses demonstrated an altered organization of virus induced membrane structures in the infected cell when TF was not produced. Thus, our data suggest that TF may be involved in virus particle assembly or release via generation of virus-induced cytopathic vacuoles. To attempt to elucidate a potential mechanism for the alteration in membrane rearrangement upon TF synthesis, we interrogated intracellular Ca2+ levels given that perturbation of ER calcium stores can lead to vesiculation of the cellular ER and that TF appears involved in virus-induced vesicle formation. Indeed, a TF knockout mutant demonstrated statistically significant lower [Ca2+]i than the wild type in mammalian and insect cells.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Biology|Virology

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