A Two-Phase Innate Host Response to Alphavirus Infection Identified by mRNP-Tagging In Vivo

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Abstract
A concept fundamental to viral pathogenesis is that infection induces specific changes within the host cell, within specific tissues, or within the entire animal. These changes are reflected in a cascade of altered transcription patterns evident during infection. However, elucidation of this cascade in vivo has been limited by a general inability to distinguish changes occurring in the minority of infected cells from those in surrounding uninfected cells. To circumvent this inherent limitation of traditional gene expression profiling methods, an innovative mRNP-tagging technique was implemented to isolate host mRNA specifically from infected cells in vitro as well as in vivo following Venezuelan equine encephalitis virus (VEE) infection. This technique facilitated a direct characterization of the host defense response specifically within the first cells infected with VEE, while simultaneous total RNA analysis assessed the collective response of both the infected and uninfected cells. The result was a unique, multifaceted profile of the early response to VEE infection in primary dendritic cells, as well as in the draining lymph node, the initially targeted tissue in the mouse model. A dynamic environment of complex interactions was revealed, and suggested a two-step innate response in which activation of a subset of host genes in infected cells subsequently leads to activation of the surrounding uninfected cells. Our findings suggest that the application of viral mRNP-tagging systems, as introduced here, will facilitate a much more detailed understanding of the highly coordinated host response to infectious agents. A major element of viral pathogenesis is the induction of specific changes within the infected host, often reflected in altered gene expression patterns. However, revealing these changes in vivo has been limited by an inability to distinguish changes within the minority of infected cells from that in surrounding uninfected cells. Here we introduce a viral mRNP-tagging system, based on Venezuelan equine encephalitis virus (VEE), that enables the isolation of host mRNA specifically from infected cells in vitro and in vivo, even when they are a small minority. This system allowed us for the first time to monitor the innate response specifically within the cells initially infected in vivo. In combination with simultaneous analysis of the entire tissue response, the result was a multifaceted view of the innate response to VEE in dendritic cells, and in the draining lymph node. The results supported a two-step response in which activation of host genes within infected cells leads to activation of bystander cells, offering insight into the process by which the greater innate immune response to alphaviruses is established in vivo. This system may be employed for a wide variety of pathogens, offering broader implications to the manner in which interactions between pathogens and their hosts are studied.

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