Analysis of Virion-Incorporated Host Proteins Required for Herpes Simplex Virus Type 1 Infection through a RNA Interference Screen

et al. (2013) Analysis of Virion-Incorporated Host Proteins Required for Herpes Simplex Virus Type 1 Infection through a RNA Interference Screen. PLoS ONE 8(1): e53276. doi:10.1371/journal.pone.0053276 Analysis of Virion-Incorporated Host Proteins Required for Herpes Simplex Virus Type 1 Infection through a RNA Interference Screen Camille Stegen. 0 Yordanka Yakova. 0 Daniel Henaff 0 Julien Nadjar 0 Johanne Duron 0 Roger Lippe 0 Karen L. Mossman, McMaster University, Canada 0 Department of Pathology and Cell Biology, University of Montreal , Montreal, Quebec , Canada Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle. To this end, we performed a small interfering RNA functional screen and found that 15 of these host proteins altered HSV-1 proliferation in cell culture, without any significant effect on cell viability. Moreover, the siRNA used had no negative consequences for Adenovirus type 5 propagation (with one exception) indicating that the modulation was specific for HSV1 and not merely due to unhealthy cells. The positive host proteins include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Remarkably, in most cases when virions were depleted for one of the above proteins, they replicated more poorly in subsequent infections in wild type cells. This highlights for the first time that both the cellular and virion-associated pools of many of these proteins actively contribute to viral propagation. Altogether, these findings underscore the power and biological relevance of combining proteomics and RNA interference to identify novel host-pathogen interactions. - Funding: This work was funded by a grant from the Canadian Institutes of Health Research (MOP 82921). YY was supported by a Canada Frederick Banting and Charles Best studentship from the CIHR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Herpes simplex virus type 1 (HSV-1) virions are composed of a DNA core within an icosahedral capsid surrounded by a heterogeneous and poorly characterized layer of proteins called tegument, which is itself wrapped in an envelope. Many of the tegument components are critical at an early stage of the infection. For example, the binding of incoming viral capsids to microtubules and their transport to the nucleus are dependent on components of the tegument, including the viral proteins UL36 and UL37 [1,2,3,4]. Furthermore, the incoming virion host shut off protein (Vhs; UL41) quickly down regulates the expression of several host proteins following viral entry [5,6] while VP16, also a tegument protein, regulates the impending cascade of viral gene expression [7]. Interestingly, two other transactivators, namely ICP0 and ICP4, have also been reported in the viral tegument and may play an early role upon entry of the incoming virus [8]. In principle, the incorporation of these molecules should be beneficial to the virus to facilitate the next round of infection. The importance and complexity of the HSV-1 tegument is illustrated by a recent mass spectrometry study of highly purified extracellular virions, which revealed they contain 23 potential viral teguments and up to 49 distinct cellular proteins [9]. This analysis showed that roughly half of the host proteins found in HSV-1 virions are proteins that had not yet been reported in any herpesviruses. In contrast, the presence of members of the annexin and heat shock protein families as well as cyclophilin A, DDX3X and components of the cytoskeleton have been documented in other Herpesviridae [9,10,11,12,13,14,15,16,17,18,19,20], suggesting a common function for these proteins. Moreover, host proteins have been documented in numerous other viral particles, including human immunodeficiency virions (HIV [21,22,23]), influenza [24], vesicular stomatitis virus [25] and vaccinia [26]. However their biological relevance is, at best, unclear. The incorporation of host proteins within mature viral particles presumably benefits the virus and may for example jump-start its replication cycle. In contrast, viruses also avoid the incorporation of proteins that can inhibit their replication. One such case is the HIV protein Vif that binds to the host APOBEC3G protein to prevent its inclusion into nascent virions and prevent deamination of the viral genome by this RNA editing protein [21]. It is thus likely that the incorporation of host proteins in viruses is not random but rather a regulated process. Unfortunately, few of these studies have examined the relevance of this phenomenon in the course of an infection [23,26,27,28,29]. In the present study, we aimed to identify amidst the 49 host proteins found in HSV-1 mature particles those that influence its replication and proliferation. To this end, we designed and validated a functional screening assay using small interfering RNAs (siRNA). We now report that 15 of these proteins have a significant impact on HSV-1 propagation in cell culture, with limited siRNA-associated toxicity or effect on the propagation of another double-stranded DNA virus, the human Adenovirus type 5. Positive hits include proteins involved in vesicular transport, gene regulation, signaling and immunity. Furthermore, we show functional evidence that the incorporation of most of these proteins within mature virions is biologically relevant for HSV-1 infectivity. Materials and Methods Cells and viruses 143B tk2 (ATCC CRL8303) and Vero (ATCC CCL81) cells were grown in Dulbeccos modified Eagles medium (SigmaAldrich) supplemented with 10% fetal calf serum (FCS, HyClone) and 2 mM L-glutamine (Invitrogen) in 5% CO2. 143B cells were also supplemented with 15 mg/ml 5-bromo-2 deoxyuridine (BrdU; Sigma) except prior to transfection and infection. The previously described HSV-1 K26GFP mutant (strain KOS) has been provided by Prashant Desai [30]. All viruses were propagated on BHK cells and titrated on Vero cells as previously described. Finally, the pIX-DE3 EGFP human Adenovirus type 5 [31] was propagated on 293 cells. Antibodies Primary antibodies were provided and diluted as follows: the anti-DDX3 R648 (1:1000) was a kind gift from Dr A. Patel [32], while anti-VP16 (1:1000) and the Remus V anti-HSV-1 antibodies were kindly provided by Dr H. Browne and Dr B. Sodeik respectively. Other a (...truncated)