Herpes Simplex Virus 2 Counteracts Neurite Outgrowth Repulsion during Infection in a Nerve Growth Factor-Dependent Manner

Abstract

During primary infection, herpes simplex virus type 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors, attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesised that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis we exposed sensory neurons to nerve growth factor (NGF) and mock or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secreted factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduced this repelling phenotype, increasing neurite numbers. The HSV-2 mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that recombinant, soluble glycoprotein G of HSV-2 (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection had not been investigated. Therefore, we addressed whether gG2 contributed to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of non-neuronal cells reduced their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2 mediated pathogenesis. Importance: Herpes simplex virus type 2 (HSV-2) is a prevalent human pathogen that establishes life-long latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that non-neuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition, and rather stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.

Identification of the Cleavage Domain within Glycoprotein G of Herpes Simplex Virus Type 2

“…To check whether the processing of SgG2 in infected cells was comparable to our transfection system, we infected human immortalised keratinocyte (HaCat) cells with an HSV-2 reporter virus of the MS strain (MS-CheGL, [ 12 ]) and detected SgG2 in cell lysates and supernatants of these cultures using a polyclonal rabbit serum, raised against two SgG2 peptides [ 12 ]. SgG2 processing in infected cells was very efficient, since we could not detect a signal of the precursor in our blot conditions ( Figure 8 D).…”

“…The generation and characterization of HSV2(MS)Lox, HSV2(MS)Lox-pHCMVmCheLuc (termed MS-CheGL in this report), mCherry-gLuc reporter virus used for infection experiments is based on the HSV-2 MS BAC strain. The construction and characterisation of these viruses are described in [ 12 ].…”

“…Blots were incubated in 3% Milk (in PBS-T) using dilutions of 1:2.000 (V5), 1:5000 (HA), 1:1000 (RFP), 1:5000 (gp120). The anti-SgG2 polyclonal rabbit serum was generated previously in our lab [ 12 ] and used at a 1:2,000 dilution in the Western blotting assays.…”

“…For infection experiments, 5 × 10 5 HaCat cells/well were seeded one day prior to infection in a 12-well plate. HSV-2 virus (MS-CheGL, [ 12 ]) inoculum was then added to reach a multiplicity of infection of 1 in a volume of 500 µl in normal growth medium. After 1 h of incubation, the inoculum was removed (3x wash with PBS) and 1 mL fresh medium was added.…”

“…Both gG1 and gG2 also bind and enhance chemokine activity as structural proteins in the viral envelope [ 10 ]. They also interact with nerve growth factor (NGF), but only SgG2 enhances NGF-mediated neurite outgrowth [ 11 , 12 ]. The enhancement of chemokine and NGF activities by SgG2 may play a role during HSV-2 infection and pathogenesis.…”