Influenza virus-host interactome screen as a platform for antiviral drug development

Abstract

Host factors required for viral replication are ideal drug targets because they are less likely than viral proteins to mutate under drug-mediated selective pressure. Although genome-wide screens have identified host proteins involved in influenza virus replication, limited mechanistic understanding of how these factors affect influenza has hindered potential drug development. We conducted a systematic analysis to identify and validate host factors that associate with influenza virus proteins and affect viral replication. After identifying over 1,000 host factors that coimmunoprecipitate with specific viral proteins, we generated a network of virus-host protein interactions based on the stage of the viral life cycle affected upon host factor downregulation. Using compounds that inhibit these host factors, we validated several proteins, notably Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) and JAK1, as potential antiviral drug targets. Thus, virus-host interactome screens are powerful strategies to identify targetable host factors and guide antiviral drug development.

Figure 1. Overview of a systematic study to elucidate the physical and functional host-viral interactions in influenza virus replication, and to identify antiviral drugs

(A, B) Schematic diagram of the identification of host proteins that co-precipitated with 11 influenza A viral proteins and affected viral replication. (A) Mass spectrometry analysis identified 1,292 host proteins that co-immunoprecipitated with one or more of the 11 FLAG-tagged influenza viral proteins. (B) To identify host factors that affect virus replication, cells were transfected with siRNAs targeted to each of the 1,292 candidate host genes and were then infected with influenza virus. Virus titers and cell viability were then determined. We identified 323 host genes whose mRNA levels were down-regulated, while virus titers were reduced by more than two log₁₀ units compared with a control (299 host factors) or increased by more than one log₁₀ unit (24 host factors). (C) To better understand the role of the identified host factors, we performed mechanistic studies assessing different steps in the viral life cycle for our ‘top hits’, that is, 91 host factors whose siRNA-mediated down-regulation reduced viral replication in cultured cells by at least three log₁₀ units while retaining >80% cell viability. (D) To identify antiviral drugs for influenza virus, we searched for drugs targeting the 299 host factors identified here and selected 11 drugs for in vitro testing. See also Figures S1–4 and Tables S1–3.

Figure 2. Network of host-influenza viral protein interactions

Interactions among the viral proteins and the 323 host factors identified here (gray and magenta circles) were visualized by using Cytoscape (http://cytoscape.org/). ‘Top hits’ (for a definition, see text and legend to Figure 1) are shown in magenta. Also shown are the steps in the viral life cycle affected by down-regulation of the respective host factor. The network image is fully-zoomable on the monitor. See also Tables S2 and S5.

Figure 3. Effects of siRNA-mediated down-regulation of the 91 ‘top hits’ on the influenza virus life cycle

A summary of the effects of siRNA-mediated down-regulation of the 91 ‘top hits’ on influenza virus replication steps. The percentages indicate the relative efficiency compared with the negative control and correspond to the values presented in Table S5. For factors with a significant effect on the early steps in the viral life cycle, polymerase activity, or VLP formation, we did not test the efficiency of vRNA and NP virion incorporation; these factors are shown in gray in the respective columns.

Figure 4. Effects of selected siRNAs targeting the 91 ‘top hits’ on the intracellular localization of viral proteins in infected cells

To examine whether the down-regulation of the 91 ‘top hits’ affects the intracellular localization of the viral proteins in virus-infected cells, siRNA-transfected HEK 293 cells were infected with 200 pfu of WSN virus per well of a 24-well tissue culture plate, fixed at 12 h post-infection, and then stained with an anti-HA, anti-NA, anti-NP, or anti-M1 antibody. The intracellular localization of HA (A), NA (B), and NP (C) is shown. None of the siRNAs affected M1 localization. See also Figure S4 and Table S5.

Figure 5. Putative roles of identified host factors in the influenza virus life cycle

Influenza virus is internalized by receptor-mediated endocytosis. The viral ribonucleoprotein (vRNP) complexes containing the 8 viral genome RNAs (depicted by orange bars) are transported into the nucleus where replication and transcription of the viral genome take place. vRNPs formed with newly synthesized viral RNA, NP, and viral polymerase proteins are transported from the nucleus to the cytoplasm. HA and NA are processed posttranslationally during their transport from the ER to the Golgi apparatus. In the late stage of the viral life cycle, virion components are transported to the plasma membrane and progeny viruses then bud from the cells. The light orange boxes indicate individual steps of the influenza virus life cycle; the gray boxes indicate host cellular processes that are likely involved. Host factors identified in this study are grouped according to the viral life cycle steps they affected; light green circles indicate host factors identified in previous studies. Among the ‘Known host factors’, only XPO1 (also known as CRM1) was identified in this study. See also Table S5.

Figure 6. Effects of selected drugs on virus titers and cell viability in virus-infected cells

HEK 293 (A) or A549 (B) cells were infected with WSN virus at an MOI of 0.001. One hour later, cells were washed and incubated with medium containing the indicated concentration of golgicide A (A) and ruxolitinib (B). DMSO (final concentration, 1%) was used as a control. Forty-eight hours later, culture supernatants were harvested for virus titration and cell viability was determined by using CellTiter-Glo. Average and standard deviation of 3 replicates are shown. The p value was calculated with Welch’s t-test compared with a non-targeting siRNA control. To control for the multiplicity effect, p values were adjusted using Benjamini-Hochberg’s procedure keeping the false discovery ratio < 0.05. Asterisk indicates that the adjusted p value is < 0.05. See also Figure S6 and Table S6.

Figure 7. Role of JAK1 in influenza virus replication

(A) siRNA targeting JAK1 reduced the formation of virus particles from virus-infected cells. HEK 293 cells transfected with the indicated siRNAs were infected with WSN virus at an MOI of 5. At 12 h post-infection, cells were subjected to transmission electron microscopy. (B) Depletion of JAK1 inhibited vRNP incorporation into virions. siRNA directed at JAK1 reduced relative levels of NP and vRNA in virions released from HEK 293 cells infected with WSN virus. See also Table S5C.