Viral pathogens are reliant on their hosts for replication. Thus, viruses have evolved many strategies to hijack and re-configure host cell processes for viral replication, transport, and dissemination. This dissertation focuses on baculoviruses, which are large DNA viruses that infect insects. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects lepidopteran insects and has a striking dependence on the host cell actin cytoskeleton, a system of filaments that function in cell shape, movement, and intracellular transport. AcMNPV hijacks the actin cytoskeleton at almost every stage of infection, utilizing it for movement and cell-cell spread. During the delayed-early stage of AcMNPV infection, cells accumulate filamentous actin at their periphery. The AcMNPV protein actin rearrangement inducing factor-1 (ARIF-1) was previously shown to be necessary and sufficient for assembly of this peripheral actin. Furthermore, baculoviruses lacking functional ARIF-1 were shown to experience a significant delay in the infection of insect organ systems and host insect death, indicating that ARIF-1 accelerates the spread of viral infection. However, the mechanisms by which ARIF-1 induces actin accumulation and accelerates viral spread remain unknown.
My dissertation research has provided insights into the molecular mechanism of ARIF-1-induced peripheral actin assembly, as well as how it might accelerate systemic viral spread in the host insect. I found that ARIF-1 induces the formation of actin structures that behave like, and have a similar composition to podosomes and invadopodia (collectively known as ‘invadosomes’) in mammalian cells. Invadosomes are protrusive structures that direct degradation of the extracellular matrix (ECM) in conjunction with cell motility. By imaging cultured insect cells expressing GFP-tagged actin, I observed that beginning at 3 h post infection and persisting for hours thereafter, invadosome-like structures form in infected cells and are arranged into clusters or rings that dynamically change shape. Furthermore, I found that actin, ARIF-1, and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to these invadosome-like structures. Finally, invadosome-like structure formation requires Arp2/3 complex activity. This indicates that invadosome-like structures in infected insect cells resemble mammalian invadosomes in their dynamics and composition. I speculate that in infected insects, ARIF-1-induced invadosome-like structures may degrade barriers to infection such as the midgut basal lamina (BL), a layer of ECM that sequesters the midgut. Degradation of the BL would allow AcMNPV to escape the midgut and spread through the insect body.
To determine how ARIF-1 induces changes in the actin cytoskeleton, I identified regions and sequences within ARIF-1 that are required for the formation of invadosome-like structures. ARIF-1 contains an N-terminal region with three transmembrane domains and a C-terminal cytoplasmic region. By constructing N-terminal and C-terminal truncations of ARIF-1, I found that the ARIF-1 C-terminal cytoplasmic region is required for formation of clusters of invadosome-like structures. Moreover, the N-terminal domain is dispensable, and the ARIF-1 C-terminal region (ARIF-1(303-417)), anchored to the plasma membrane by a heterologous transmembrane domain, is sufficient for formation of clusters of invadosome-like structures. Additionally, I mapped the residues required for ARIF-1 function by mutating seven individual tyrosine residues and eleven individual proline residues in the ARIF-1 C-terminal cytoplasmic region. I found that ARIF-1 residues Y332 and P335 play a role in forming clusters of invadosome-like structures. I speculate that Y332 is phosphorylated, and both Y332 and P335 serve as binding sites for host proteins that regulate actin assembly. Recruitment of these host proteins to ARIF-1 likely culminates in activation of the Arp2/3 complex and invadosome assembly/clustering. Future research will reveal the details of ARIF-1 induced invadosome-like structure formation, as well as if these structures function as invadosomes in the context of insect infection.
