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

Abstract

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 HSV-1 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.

The ATP-Dependent RNA Helicase DDX3X Modulates Herpes Simplex Virus 1 Gene Expression

“…Furthermore, the GFP-tagged viral particles often, but not always, colocalized with DDX3X in the cytoplasm (Fig. 5B), in agreement with the incorporation of this cellular protein in mature virions (21). A time course revealed that DDX3X started to aggregate between 3 and 6 h postinfection (hpi) and that this intensified with time (Fig.…”

“…This confirmed that DDX3X is indeed a modulator of the HSV-1 life cycle. It also ruled out that nonspecific off-target effects were responsible for our past findings (21).…”

“…We previously reported by proteomics that HSV-1 incorporates the host protein DDX3X in mature virions (16) and that both the cellular and virion-incorporated pools of DDX3X influence viral yields (21). To independently validate the role of the cellular DDX3X in viral replication, we resorted to the BHK21-derived tsET24 cell line, which harbors a temperature-sensitive (ts) DDX3X inactive at 39.5°C but functional at 34°C (22).…”

“…Most interestingly, depleting either the viral or the cellular pool of DDX3X significantly impairs HSV-1 infectivity (21). As the exact function of DDX3X in the HSV-1 replication cycle is not clear, we examined more closely the nature of this host-pathogen interaction.…”

The Tegument Protein pp65 of Human Cytomegalovirus Acts as an Optional Scaffold Protein That Optimizes Protein Uploading into Viral Particles

“…These proteins are known to be involved in the regulation of signal transduction pathways by modulating the activities of client proteins via interaction with phosphoserine or phosphothreonine. Interestingly, Stegen and colleagues recently showed in a small interfering RNA (siRNA) screen that one of the 14-3-3 family members, 14-3-3 zeta, contained in virions, significantly supported HSV-1 production in cell culture (62). It remains to be determined if 14-3-3 proteins also influence HCMV infection.…”