Herpes Simplex Virus 1 Strains 17 syn+ and KOS(M) Differ Greatly in Their Ability To Reactivate from Human Neurons In Vitro

Abstract

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in peripheral nerve ganglia. Periodically, the virus reactivates from this latent reservoir and is transported to the original site of infection. Strains of HSV-1 have been noted to vary greatly in their virulence and reactivation efficiencies in animal models. While HSV-1 strain 17syn⁺ can be readily reactivated, strain KOS(M) shows little to no reactivation in the mouse and rabbit models of induced reactivation. Additionally, 17syn⁺ is markedly more virulent in vivo than KOS. This has raised questions regarding potential strain-specific differences in neuroinvasion and neurovirulence and their contribution to differences in the establishment of latency (or ability to spread back to the periphery) and to the reactivation phenotype. To determine if any difference in the ability to reactivate between strains 17syn⁺ and KOS(M) is manifest at the level of neurons, we utilized a recently characterized human neuronal cell line model of HSV latency and reactivation (LUHMES). We found that KOS(M) established latency with a higher number of viral genomes than strain 17syn⁺ Strikingly, we show that the KOS(M) viral genomes have a higher burden of heterochromatin marks than strain 17syn⁺ The increased heterochromatin profile for KOS(M) correlates with the reduced expression of viral lytic transcripts during latency and impaired induced reactivation compared to that of 17syn⁺ These results suggest that genomes entering neurons from HSV-1 infections with strain KOS(M) are more prone to rapid heterochromatinization than those of 17syn⁺ and that this results in a reduced ability to reactivate from latency.IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes a lifelong infection in neuronal cells. The virus periodically reactivates and causes recurrent disease. Strains of HSV-1 vary greatly in their virulence and potential to reactivate in animal models. Although these differences are phenotypically well defined, factors contributing to the strains' abilities to reactivate are largely unknown. We utilized a human neuronal cell line model of HSV latency and reactivation (LUHMES) to characterize the latent infection of two HSV-1 wild-type strains. We find that strain-specific differences in reactivation are recapitulated in LUHMES. Additionally, these differences correlate with the degree of heterochromatinization of the latent genomes. Our data suggest that the epigenetic state of the viral genome is an important determinant of reactivation that varies in a strain-specific manner. This work also shows the first evidence of strain-specific differences in reactivation outside the context of the whole animal at a human neuronal cell level.

Keywords: herpes simplex virus; latency; neurotropism; neurovirulence; reactivation.

FIG 1

Experimental timeline of neuronal differentiation, infection, latency, and reactivation. (A) Experimental timeline of acute infection. LUHMES neuronal cells were plated and allowed to proliferate for 3 days, followed by 5 days of differentiation, as described in Materials and Methods. Once differentiated, cells were infected with either 17syn⁺ or KOS(M) at an MOI of 5 for 1 h, with harvesting of DNA/RNA at the indicated times. (B) Experimental timeline of latency and reactivation. LUHMES neuronal cells were plated and allowed to proliferate for 3 days, followed by 5 days of differentiation. Once differentiated, cells were pretreated for 2 h in medium containing 50 μM acyclovir (ACV). Following pretreatment with ACV, cells were infected with either 17syn⁺ or KOS(M) at an MOI of 5 in medium containing 50 μM ACV. Forty-eight hours later, the medium was changed to medium without ACV and the infection was allowed to proceed, with harvesting at the latent time point 8 days postinfection (labeled 0 hours postreactivation). Reactivation was induced at day 8 postinfection (labeled as day 16) with 10 μM final concentration of Ly294002, and reactivation samples were harvested at the indicated times.

FIG 2

HSV-1 genome copies during acute infection. (A) HSV-1 strain KOS(M) delivers and maintains significantly higher genome copies than 17syn⁺ during the acute infection of LUHMES neurons. HSV-1 genome copy numbers were tracked by qPCR using primers and probes designed against UL30 over a 24-h time course of infection of LUHMES cells with either 17syn⁺ or KOS(M) at an MOI of 5. (B) HSV-1 strain 17syn⁺ genome copies were determined as described above. A significant difference in genome copies was seen 12 and 24 h postinfection compared to levels at 0 h. Interestingly, at 12 h postinfection, genomic copies were reduced compared to those at 0 h, and input genomes appear to have replicated by 24 h postinfection. (C) HSV-1 strain KOS(M) input genomes undergo statistically signification replication by 24 h postinfection compared to levels at 0 h. For each time point, significance was determined by ordinary two-way ANOVA with Sidak’s multiple-comparison test across 6 biological replicates (*, P < 0.05; ***, P < 0.0001) (n = 6).

FIG 3

More transcription is occurring per genome for strain 17syn+ than KOS(M) during acute infection of LUHMES. Shown are the relative gene expression profiles for HSV-1 ICP4 (A), TK (B), and LAT intron (C) expressed as copies per genome following a 24-h time course of infection of LUHMES with 17syn⁺ or KOS(M) at an MOI of 5. RT-qPCR was performed with primers and probes against the indicated genes (Table 2). Interestingly, on a per-genome basis, higher gene expression is seen for all three transcripts measured when LUHMES cells are infected with 17syn⁺ versus KOS(M). For each time point, significance was determined by ordinary two-way ANOVA with Sidak’s multiple-comparison test across 6 biological replicates (*, P < 0.05; ***, P < 0.0001) (n = 6).

FIG 4

Time course plaque assay during establishment of latent infection. KOS(M) appears to enter latency earlier than 17syn⁺, as measured by less infectious virus production during the course of latency establishment. The data are represented as the number of infectious particles per LUHMES culture containing 150,000 cells. For each time point, significance was determined by ordinary two-way ANOVA with Sidak’s multiple-comparison test across 12 biological replicates (**, P < 0.01) (n = 12).

FIG 5

KOS(M) genomes are maintained at higher copies overall during latency and reactivation than with 17syn⁺ but are not significantly replicated following PI3K inhibitor-induced reactivation. (A) HSV-1 strain KOS(M) maintains significantly higher genome copies than 17syn⁺ during latency as well as during PI3K inhibitor-induced reactivation in LUHMES. Reactivation was induced at day 8 postinfection (0 h) with 10 μM Ly294002, and cultures were harvested for qPCR at the indicated times using primers and probes against UL30. (B) 17syn⁺ genome copies have been significantly replicated by 12 h postreactivation (compared to 0 h). (C) HSV-1 strain KOS(M) genome copies were determined as described above. We found no significant difference in genomes copy number during reactivation compared to that at 0 h, implying fewer genomes were replicated. For each time point, significance was determined by ordinary two-way ANOVA with Sidak’s multiple-comparison test across 6 biological replicates (*, P < 0.05; ***, P < 0.0001) (n = 6).

FIG 6

Transcripts produced per genome during the latent infection and following PI3K inhibitor-induced reactivation differ between 17syn⁺ and KOS(M). Relative ICP4 (A), TK (B), LAT intron (C), and US3 (D) expression calculated during latency and following reactivation are expressed on a per-genome basis. Reactivation was induced at day 8 postinfection with 10 μM Ly294002, and reactivation samples were harvested at the indicated times. RT-qPCR was performed as described in Materials and Methods and Table 2. For each time point, significance was determined by ordinary two-way ANOVA with Sidak’s multiple-comparison test across 6 to 12 biological replicates (*, P < 0.05; **, P < 0.001; ***, P < 0.0001) (n = 6 to 12).

FIG 7

HSV-1 strain KOS has increased H3K27me3 marks compared to those of strain 17syn⁺ during latency and PI3K inhibitor-induced reactivation in LUHMES. Chromatin immunoprecipitation was performed to measure heterochromatin formation on HSV-1 genomes from cultures of LUHMES cells infected with 17syn⁺ or KOS(M) at 8 days postinfection (latency) or following Ly294002-induced reactivation from latency. Shown are three regions of the HSV-1 genome queried by qPCR following ChIP. (A to C) ICP4 (A), the LAT promoter (B), and the late gene, gC (C). Heterochromatin was assessed by immunoprecipitation with an antibody recognizing H3K27me3. ChIP-qPCR data were normalized to percent input over total H3. Reactivation was induced at day 8 postinfection with 10 μM Ly294002, and reactivation samples were harvested at the indicated times. These data point to a significant reason why KOS(M) fails to reactivate. As shown here, 17syn⁺ genomes are associated with fewer heterochromatin marks than KOS(M), indicating less accessibility of the KOS genome for replication and transcriptional activities. For each time point, significance was determined by ordinary two-way ANOVA with Sidak’s multiple-comparison test across 3 biological replicates (*, P < 0.05; **, P < 0.001; ***, P < 0.0001) (n = 3).