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

Abstract

The human protein DDX3X is a DEAD box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, and mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication, and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection, but, rather, the protein controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway. IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, an RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export, and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here, we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and, consequently, virion assembly by a process that is independent of its ability to promote the interferon pathway. KEYWORDS DDX3X, helicase, herpes, host-pathogen interaction, DNA virus, RNA virus, interferon, herpes simplex virus, host-pathogen interactions, transcriptional regulation, translational control T he human DDX3 protein is a member of a large family of DEAD box ATP-dependent RNA helicases. In humans, it is encoded by the X (DDX3X) and Y (DDX3Y) chromosomes, albeit the latter is restricted to testes (1). It participates in different stages of cellular gene expression, such as transcription, mRNA maturation, and mRNA export and translation (2). Given these crucial roles in RNA biology, several RNA viruses interact with DDX3X, often with important consequences for viral replication. This includes hepatitis C virus (HCV), norovirus, West Nile virus, and Japanese encephalitis virus (3-7). This is also the case for HIV and hepatitis B virus (HBV), a peculiar DNA virus that relies on an RNA template and reversed transcription to replicate its genome (8,9). Furthermore, DDX3X also contributes to innate immunity against these viruses. For instance, DDX3X stimulates interferon (IFN) type I production by binding IKK (IB kinase epsilon) and TBK1 (tank-binding kinase 1), leading to IRF3 phosphorylation and activation (10,

Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles

“…Depletion of VP16 or VP22 in virions hints at a complex scenario. To orthogonally confirm our findings, we used an RNA interference strategy to lower the amounts of the proteins in the virions and assessed their infectivity, an approach we successfully used in the past for VP16 and several host proteins incorporated in virions (13,26). As anticipated, short interfering RNA (siRNA) targeting VP16 or VP22 significantly reduced the level of expression of these proteins in infected cell lysates (Fig.…”

Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences

“…Thus, removing the region interferes with protein expression . This hypothesis would also be consistent with the stimulation of HSV TK gene expression by the cellular RNA helicase DDX3X . These assumptions are not in contradiction to the lack of reactivity of the LS +5/+15 mutant in HSV‐infected mouse L cells as the study only looked at TK mRNA not the protein levels .…”

“…Thus, proteins binding to the region +5 to +15 of the 5 0 -UTR-coding DNA might not be important for the regulation of HSV TK expression. Recently, it was shown that the cellular RNA helicase DDX3X is important for the regulation of HSV early genes including UL23, which encodes for HSV TK, suggesting that TK mRNA structures or small RNA may regulate HSV TK gene expression in HSV-infected cells [52]. It is important to note that none of the cell lines studied here expresses ICP4 and the putative factor binding to the region +5 to +27 of the 5 0 -UTR at the DNA or RNA level seems to act as a repressor and not an activator in uninfected mouse and human cells as removing its putative binding site increases protein expression.…”

Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

“…Wild-type HSV-1 strain 17 ϩ and the HSV-1 (17 ϩ ) Lox-Luc viral strain (both obtained from Beate Sodeik) were propagated on BHK cells, and their titers were determined by plaque assay on Vero cells. The latter virus is derived from strain 17 ϩ and encodes a Gaussian luciferase gene under the constitutively expressed immediate-early cytomegalovirus (CMV) promoter positioned between the UL55 and UL56 HSV-1 genes (57).…”