Mutational analysis of the herpes simplex virus type 1 ICP0 C3HC4 zinc ring finger reveals a requirement for ICP0 in the expression of the essential alpha27 gene

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It targets several host factors for proteasomal degradation and subsequently activates viral expression. ICP0 has a nuclear localization sequence and functions in the nucleus early during infection. However, later in infection, ICP0 is found solely in the cytoplasm. The molecular mechanism and biological function of the ICP0 nuclear-to-cytoplasmic translocation are not well understood. In this study, we sought to characterize elements important for this translocation. We found that (i) in human embryonic lung fibroblast (HEL) cells, ICP0 C-terminal residues 741 to 775 were necessary but not sufficient for the nuclear-to-cytoplasmic translocation; (ii) the loss of ICP0 E3 ubiquitin ligase activity, which led to defective viral replication in nonpermissive cells, also caused mutant ICP0 to be retained in the nucleus of HEL cells; (iii) in permissive U2OS cells, however, ICP0 lacking E3 ligase activity was translocated to the cytoplasm at a pace faster than that of wild-type ICP0, suggesting that nuclear retention of ICP0 occurs in an ICP0 E3 ligase-dependent manner; and (iv) the ICP0 C terminus and late viral proteins cooperate in order to overcome nuclear retention and stimulate ICP0 cytoplasmic translocation. Taken together, less ICP0 nuclear retention may contribute to the permissiveness of U2OS cells to HSV-1 in the absence of functional ICP0. A distinct characteristic for eukaryotes is the compartmentalization of cell metabolic pathways, which allows greater efficiency and specificity of cellular functions. ICP0 of HSV-1 is a multifunctional viral protein that travels through different compartments as infection progresses. Its main regulatory functions are carried out in the nucleus, but it is translocated to the cytoplasm late during HSV-1 infection. To understand the biological significance of cytoplasmic ICP0 in HSV-1 infection, we investigated the potential players involved in this nuclear-to-cytoplasmic translocation. We found that there is a nuclear retention force in an ICP0 E3 ubiquitin ligase-dependent manner. In addition, we identified the C terminus of ICP0 as a element cooperating with late viral proteins to overcome the nuclear retention and stimulate the nuclear-to-cytoplasmic translocation of ICP0.

Section: Resultsmentioning

confidence: 99%

Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection

Samrat

Zheng

et al. 2018

“…Alternatively, the reduced ICP4 and ICP27 mRNA levels in the presence of the LATs might be a consequence of reduced ICP0 mRNA levels. Indeed, it was shown that, in the absence of structural viral proteins such as Vmw65 (10) and at a low MOI, ICP0 can activate the expression of ICP4 and ICP27 genes (10,30,36). Further experiments are in progress to address the mechanism of action of the LATs upon ICP0, ICP4, and ICP27.…”

Section: Discussionmentioning

confidence: 99%

“…We chose initially to examine the influence of the LATs on ICP0 expression for the following reasons. (i) ICP0 is the first IE gene to be expressed at very early times postinfection (36). (ii) This IE protein is a transactivator which enhances HSV-1 replication during acute infection as well as reactivation from latency (9) and is capable of activating all three kinetic classes of HSV genes (10).…”

Section: ͻ4 ͻ4mentioning

confidence: 99%

Herpes Simplex Virus Type 1 Latency-Associated Transcripts Suppress Viral Replication and Reduce Immediate-Early Gene mRNA Levels in a Neuronal Cell Line

Mador¹,

Goldenberg²,

Cohen³

et al. 1998

During herpes simplex virus type 1 (HSV-1) latent infection in human dorsal root ganglia, limited viral transcription, which has been linked to HSV-1 reactivation ability, takes place. To study the involvement of this transcription in HSV-1 replication in neuronal cells and consequently in viral latency, we constructed stably transfected neuronal cell lines containing (i) the entire HSV-1 latency transcriptionally active DNA fragment, (ii) the same DNA sequence with deletions of the latency-associated transcript (LAT) promoters, or (iii) the DNA coding sequence of the LAT domain. Replication of HSV-1 or a LAT-negative mutant was markedly repressed in the LAT-expressing cells, a phenomenon mediated by the LATs. To study the mechanism responsible for this effect, we examined LAT influence upon expression of HSV-1 immediate-early (IE) genes ICP0, ICP4, and ICP27, by Northern blot analysis. Following infection of a LAT-expressing neuronal cell line with a LAT-negative mutant, the steady-state levels of all three IE mRNAs were reduced compared to those for control cells. Transient transfections into a neuronal cell line indicated that the LAT suppressive effect upon ICP0 mRNA was mediated directly and was not due to the LAT effect upon the ICP0 promoter. We therefore propose that the LATs may repress viral replication in neuronal cells by reducing IE gene mRNA levels and thus facilitate the establishment of HSV-1 latency in nervous tissue.

“…HSV-1(F) is a limited-passage isolate described before (79). The properties of the ΔICP0, R7914, ICP0 Ring Finger (RF), ΔICP27, ΔICP8, R2621 (ΔVHS), ΔUL46, and R3616 mutant viruses were described elsewhere (35,40,(80)(81)(82)(83)(84)(85).…”

Section: Methodsmentioning

confidence: 99%

Biogenesis of Extracellular Vesicles during Herpes Simplex Virus 1 Infection: Role of the CD63 Tetraspanin

Dogrammatzis

Deschamps

Kalamvoki

2019

Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The virus primarily infects mucoepithelial cells and establishes latent reservoirs in neurons in sensory ganglia. Frequent reactivation has been linked to severe diseases, especially in immunocompromised individuals. Earlier, we reported that viral and host factors are packaged in extracellular vesicles (EVs) and delivered to uninfected cells, where they activate antiviral responses and restrict virus infection. Here, we interrogated the effect of HSV-1 infection on EV biogenesis. We found that HSV-1 infection causes a decrease in the amount of intracellular CD63 protein with a concomitant increase in extracellular CD63. This observation correlates with our previous finding that infected cells release more CD63-positive EVs than uninfected cells. The stimulation of CD63 exocytosis requires virus replication. CD63 is a member of the tetraspanin family of proteins that traffics between the plasma membrane and endosomal compartments and has a role in sorting cargo into the EVs. Previously, we reported that in cells depleted of CD63, HSV-1 virus yields increased, and here we provide data showing that in cells overexpressing CD63, HSV-1 virus yields decreased. Taken together, our data indicate that CD63 negatively impacts HSV-1 infection and that the CD63-positive EVs could control the dissemination of the virus in the host. Perhaps EV release by HSV-1-infected cells is a mechanism that controls virus dissemination. IMPORTANCE Intercellular communication, especially in neurons, largely relies on EVs, and modulation of EVs is known to impact physiological processes. Here, we present evidence that HSV-1 infection causes major alterations in the biogenesis of EVs, including an increase in their number and an increase in the CD63-positive population of EVs. These alterations result in an enrichment of the milieu of infection with EVs carrying signatures from infected cells. In addition to changes in the origin and type, EVs released by infected cells have differences in cargo, as they carry viral and host factors determined by the virus. The tetraspanin CD63 negatively impacts the infection, as demonstrated by CD63-knockdown and overexpression assays. A proposed mechanism involves the activation of antiviral responses in cells receiving CD63-positive EVs released by infected cells. Overall, HSV-1 causes major alterations in EVs that could contribute to HSV-1 persistence and pathogenesis.