Host-Pathogen Investigation

Our lab has applied genetic tools to uncover how viruses exploit mammalian proteins to further their goals of reproduction and how the cell combats these viruses. We identified a large number of host factors required for a wide variety of viruses to replicate and factors that restrain this replication. We hope that by understanding these pathways, we can inform antiviral drug discovery and host-pathogen biology. We have also initiated a chemical screening effort to explore how existing FDA approved drugs can be used in combination to control acute viral infections.

Most antiviral drugs target proteins on the virus.  While these can have efficacy, the virus has a ready mechanism through which to overcome these drugs by mutating residues involved in drug binding. However, drugs that interfere with host pathways upon which the virus relies cannot be easily overcome by the virus by mutation because it would require the virus to evolve an entirely different functionality, and extremely low probability event.  In order to identify potential anti-drug targets, we began an analysis of several important viral pathogens to identify the host factors upon which they rely with an eye toward finding anti-viral drug targets.

HIV: A Genetic Map of the HIV Life Cycle and Host Cell Dependencies

HIV-1 exploits multiple host proteins during infection. We performed the first large-scale genome-wide siRNA screen to identify host factors required by HIV-1 and identified 274 HIV-dependency factors (HDFs) (207).

These proteins participate in a broad array of cellular functions and implicate new pathways in the viral lifecycle. Further analysis revealed previously unknown roles for retrograde Golgi transport proteins (Rab6 and Vps53) in viral entry, a karyopherin (TNPO3) in viral integration, and the Mediator complex (Med28) in viral transcription. Transcriptional analysis revealed that HDF genes were enriched for high expression in immune cells suggesting that viruses evolve in host cells that optimally perform the functions required for their lifecycle.  These host factors represent potential therapeutic targets. We have mapped these functions onto a cell to show the pathways HIV must exploit to propagate.  We are currently completing this work with funding from the Gates Foundation to use all existing RNA interference methods to identify all detectible host factors needed for the HIV lifecycle.  This information will be integrated into protein interaction maps to realize an integrated view of the HIV lifecycle and possible points of intervention.

HCV: A Screen for Host Factors Needed for Viral Propagation

Hepatitis C virus (HCV) infection is a major cause of end stage liver disease and a leading indication for liver transplantation. Current therapy fails in many instances and is associated with significant side effects. HCV encodes only a few proteins and depends heavily on host factors for propagation. Each of these host dependencies is a potential therapeutic target. To find host factors required by HCV, we completed a genome-wide small interfering RNA (siRNA) screen using an infectious HCV cell culture system (232). We applied a two-part screening protocol to allow identification of host factors involved in the complete viral lifecycle.

The candidate genes found included known or previously identified factors, and also implicate many additional host cell proteins in HCV infection. To create a more comprehensive view of HCV and host cell interactions, we performed a bioinformatic meta-analysis that integrates our data with those of previous functional and proteomic studies. The identification of host factors participating in the complete HCV lifecycle will both advance our understanding of HCV pathogenesis and illuminate novel therapeutic targets.

Influenza A: Host Dependency Factors

Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses anti-viral restriction factors to defend against infection. To find host-cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors (IDFs) with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing (233).

IFITM Proteins – Mediators of Antiviral Innate Immunity

The siRNA screen also led to the discovery that the interferon-inducible trans-membrane proteins, IFITM1, 2 and 3, restrict early replication of influenza A virus (233). The IFITM proteins confer basal resistance to influenza A virus, but are also inducible by interferons (IFN) type I and II, and are critical for IFN’s virustatic actions.  Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus (DNV) and West Nile virus (WNV).

The IFITM protein family is highly conserved in evolution all the way down to fish.  While it remains to be seen if it functions to control innate immunity in these organisms, the fact that the gene family is ancient and still works today to thwart influenza A and other viruses suggests that it acts in a manner not easily overcome by these viruses and is probably attaching a part of the pathway that does not involved recognizing the virus itself, which would allow a simple mechanism of circumvention by the virus, perhaps by rerouting its transport within the cells once it has been endocytosed. Our current clues on the function of these proteins are that they are expressed on the cell surface and internally on vesicles and that they work at entry as the first line defense against viral entry.  Understanding the mechanism of their action should provide greater insight into how to combat Influenza and other viruses.  In addition, variation in the levels or activity of IFITM proteins may lead to enhanced resistance or sensitivity to infection by the flu and the severity of the infection for individuals.  Since removing IFITM proteins allows influenza A to replicate much more efficiently, we envision engineering vaccine production strains to inhibit IFITM proteins so as to speed up vaccine production.  In addition, one of the major threats to human health is the large reservoir of influenza that exists in farm animals such as swine and poultry that have a large amount of human contact.  The engineering of these animals to express higher levels of IFITM proteins could eliminate them as reservoirs of virus that then recombine with human-tropic viruses to create pandemic strains of flu.