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Based on the M3 mutant, we designed specific g-RNA sequences targeting the Us3 and Us5 genes according to the technical requirements of CRISPR–Cas9 (Supplementary Table S1). The mutated plasmids were then constructed. Based on the previously reported method (Xu et al. 2016), we first generated a deletion of the Us3 gene in the genome of M3. After modifying the sequence of the Us3 gene, the mutant M4 was cloned and isolated. Similarly, we also deleted the Us5 gene in the M4 mutant (Fig. 1A) and ultimately isolated the mutant M5 (Fig. 1A, 1B). Further sequencing results showed that the Us3 gene contained a deletion from 1248 to 1393 bp and that the Us5 gene contained a deletion from 227 to 390 bp (Fig. 1C). During the experiment, we passaged the M5 mutant for approximately 15 generations, and the deletions in the Us3 and Us5 genes were always stable during this process (results not shown). To identify the mutations in M5, based on the previously reported sequence result for the M3 strain, we not only verified the mutated target genes of UL7, UL41, LAT, and Us3/Us5 but also assessed 12 genes related to viral replication, immune evasion, and viral structure, including RS1, RL1, RL2, Us6, Us12, UL5, UL15, UL23, UL29, UL44, UL48 and UL49 (Supplementary Table S3; Supplemental file S1). The results confirmed that the target genes were mutated and the remaining genes related to viral immune evasion were unaltered (Supplemental file S1). Moreover, to explore the change in expression of the targeted proteins, we purified the mRNA of the WT, M3, M4, and M5 strains. The cDNAs were then synthesized and used to amplify the Us3 and Us5 genes. The result showed frameshift mutations in the Us3 and Us5 genes (Fig. 1D; Supplementary file S2). In addition, compared with that of the WT strain, the M3 and M5 mutants exhibited a reduced proliferation capacity in human diploid cells (Fig. 1E). These results suggest that mutations in the Us3 and Us5 genes do not affect the proliferative capacity of the HSV-1 M3 mutant.
Figure 1. Identification of the mutated genes and dynamic growth characteristics of the M5 strain in cultured cells. A Flowchart of the design and construction of the M4 and M5 viruses. B Identification of the mutated Us3 and Us5 genes in the M5 strain. The mutations were identified via PCR using specific primers. C Identification of the mutated site in the Us3 and Us5 genes in the M5 strain. D Identification of the mutated site of the mRNA in the Us3 and Us5 gene in the M5 strain. E Growth curve of the M3, M5, and WT strains in KMB17 cells. ***P < 0.001.
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A large number of studies have shown that the Us3 and Us5 genes are involved in the immune escape process during viral infection, mainly by inhibiting apoptosis induced by cytotoxic T lymphocytes (CTLs)(Aubert et al. 2006). However, HSV-1 is capable of encoding various proteins to regulate apoptosis in infected cells. The biological effect of a single protein might be compensated by other proteins. Therefore, we aimed to observe the rate of apoptosis in cells infected by the strain with mutated Us3 and Us5 to confirm the relationships of these genes with the viral capacity to inhibit cellular apoptosis as an immune evasion strategy. Cultured Jurkat human T lymphocytes were infected with the WT, M3, and M5 mutant strains at an MOI of 1. Cell apoptosis was evaluated through DAPI staining at 24 h post viral infection. In contrast to the rare apoptotic cells among the WT virus- or M3-infected cells, 10%–30% of the cells in the M5-infected groups were morphologically distinct, with cell volume contraction, chromatin condensation, and the formation of membraneenveloped apoptotic bodies in the visual field (Fig. 2A). To further test the above observations, we used Annexin V and propidium iodide (PI) to dual-label cells and to quantitatively detect the number of apoptotic cells with flow cytometric analysis. The results showed that apoptosis occurred in 4.35% and 5.04% of cells infected with the WT and M3 viruses, respectively, and in 21.31% of cells infected with M5 virus (Fig. 2B, 2E).
Figure 2. Analysis of apoptosis in Jurkat cells or HaCat cells infected with the WT, M3, or M5 strain. A, C The infected Jurkat cells or HaCat cells were stained with DAPI. The stained cells were visualized using fluorescence microscopy. Apoptotic cells show hyperfluorescence and nuclear fragmentation, and these cells are indicated with red arrows. B, E Jurkat cells were stained with Annexin V and propidium iodide (PI) at 24 h post viral infection. Flow cytometric analysis results and the percentage of Annexin V and PI-positive cells are presented. D, F HaCat cells were stained with Annexin V and PI at 8 h post viral infection. Flow cytometric analysis results and the percentage of Annexin V and PI-positive cells are presented. Jurkat or HaCat cells without viral infection were stained with Annexin V and PI as control group. **P < 0.01.
Furthermore, examining the apoptosis induction in infecting physiologically relevant mucosal cells such as HaCat keratinocytes may provide more important information about future use of the M5 strain. Cultured HaCat cells were infected with the WT, M3, and M5 mutant strains at an MOI of 1. Cell apoptosis was evaluated through DAPI staining at 8 h post viral infection. In contrast to the rare apoptotic cells among the WT virus- or M3-infected cells, few of the cells in the M5-infected groups were morphologically distinct (Fig. 2C). Moreover, we used Annexin V and PI to dual-label cells and to quantitatively detect the number of apoptotic cells with flow cytometric analysis. The results showed that apoptosis occurred in 0.65% and 0.76% of cells infected with the WT and M3 viruses, respectively, and in 2.11% of cells infected with M5 virus (Fig. 2D, 2F). These results suggested that the Us3 and Us5 genes might be involved in the inhibition of virus-related apoptosis.
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Our previous work on mice infected by the M3 strain revealed attenuated viral pathogenesis, which presented as reduced inflammatory reactions in various organs in association with a lower tissue viral load (Xu et al. 2017b). In the current work, we used the M5 strain to infect mice at a dose of 104 PFU by the intranasal route, and mock-infected control mice were established with PBS inoculation (Fig. 3A). Histopathologic examination of various organs from M5-infected mice at days 3, 7, 28, and 56 post infection indicated that all tissues, especially nervous system tissues, showed only slight infiltration of inflammatory cells without obvious pathologic lesions (Fig. 3B), which was similar to our observations in mice infected with M3 (Xu et al. 2017b). This pathologic feature was associated with asymptomatic infection until day 56 post infection. These data suggested an attenuated phenotype of M5 characterized by a non-pathologic phenotype compared with that of the WT strain. Further detection of the viral load in tissues collected from M5-infected mice indicated a significantly lower viral load in central nervous system (CNS) tissues, including the brain and trigeminal ganglion (Fig. 3C, 3D), which might lead to the observed attenuated phenotype of M5.
Figure 3. The attenuated phenotype of the M5 strain in mice. A Design of the mouse experiment for the M5 strain infection. Mice were infected with WT, M5, or mock (PBS) via the intranasal instillation of 1 × 104 PFU. B Pathological changes in the cerebrum in mice infected with WT, M5, or PBS. The tissue sections were stained with hematoxylin and eosin and imaged using an optical microscope. Tissue hyperemia and infiltration of inflammatory cells detected at 7 d.p.i. are indicated with blue and black arrows, respectively. Scale bars = 100 μm. Assessment of viral load in the brain (C) or trigeminal ganglion (D) of mice infected with WT, M5, or PBS, as determined by RT-qPCR. The viral copy numbers were quantified according to an HSV-1 DNA standard. The data are shown as the mean ± SEM based on data from three independent mice. *P < 0.05; **P < 0.01;***P < 0.001.
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Based on observations of the discrepant ratios of cellular apoptosis induced by M5 and the WT strain in Jurkat human T lymphocytes and HaCat cells, we inferred that M5 lost, to some extent, the inhibitory effect on cellular apoptosis that is usually observed during WT infection and is one of the mechanisms of viral immune evasion (Yu and He 2016). We hypothesized that M5 might present better immunogenicity than the WT and M3 strains in mice. Therefore, mice infected with M3, M5, or mock-infected (PBS) were used for further experiments, as described in Fig. 4A, in which the mice infected with M3, M5, or mockinfected (PBS) were challenged separately with the HSV-1 WT strains McKrae and 17+ at a dose of 104 PFUs through the intranasal route. The results did not reveal any visible pathological symptoms in any inoculated mice from the groups infected with the M3 and M5 strains (group: M3 and M5) until 56 days after infection. Importantly, at 28 or 56 days after M5 infection and before WT challenge, all M5-infected mice exhibited a positive serum-neutralizing antibody response with a titer of 1:4–1:8 (Fig. 4B), while the ELISpot assay with the HSV-1 membrane protein gB and ICP6 antigen stimulation showed an obvious positive IFN-γ-specific response (Fig. 4C). Further pathological observations of mice infected with M3 or M5 followed by challenge with the respective WT strains (groups: M3- McK, M3-17+, M5-McK, and M5-17+) for 56 days revealed no visible pathological symptoms in any individual (Table 1). The body weights of these mice increased normally (Fig. 4D, 4E), while most of the mice in the mock-McK and mock-17+ groups showed back arching, inverted hair, blindness, paralysis, and even death. At 28 days post challenge, all the mock-McK mice developed one or more symptoms (Table 1). Serious symptoms in mice in the mock-McK and mock-17+ groups included orbital inflammation and keratitis, and blindness (whitening of the transparent cornea and the black pupil). Moreover, the mice had a survival rate of only 12.5% (Fig. 4F). Similarly, all mock-17+ mice developed one or more of the above symptoms on the 6th day post challenge and showed a survival rate of 0% at 28 days (Fig. 4F). However, no death was found in mice infected with M5 followed by challenge with the McKrae or 17+ strains until 56 days post immunization (Fig. 4G), similar to the M3 group.
Figure 4. Immune response to HSV-1 induced in mice infected with either the HSV-1 mutant M3, the HSV-1 mutant M5, or PBS and pathological observation of WT-, M3-, and M5-infected mice after viral challenge. A Design of the mouse experiment. B The neutralizing capability of antibodies specific for HSV-1 in mice infected with HSV-1 M3, HSV-1 M5, or mock infected (PBS) (n = 3 for each). The geometric mean titers (GMTs) of neutralizing antibodies were measured by a neutralization test as described in the methods. The GMT values for the mock (PBS) groups were all < 2. C ELISpot responses to IFN-γ-secreting cells from splenic lymphocytes in HSV-1 M3- infected, HSV-1 M5-infected and mock-infected (PBS, n = 3 for each) mice. Splenic lymphocytes were incubated for 24 h in the presence of a stimulus. The positive control was phytohaemagglutinin (PHA). D–G The M3-, M5-, or mock-infected mice were challenged with WT virus at 28 (D, F) or 56 (E, G) days post viral infection. D, E The weights of the mice were measured every 2 days. F, G The morbidities of mice were observed over a 10-day period. The values are presented as the mean ± SEM. **P < 0.01; ***P < 0.001.
Pathologic symptoms 28 d.p.i. 56 d.p.i. M3-
McKM3-
17+M5-
McKM5-
17+Mock-
McKMock-
17+M3-
McKM3-
17+M5-
McKM5-
17+Mock-
McKMock-
17+Back arching 0 0 0 0 87.5 87.5 0 0 0 0 75 87.5 Inverted hair 0 0 0 0 100 87.5 0 0 0 0 75 87.5 Unilateral blindness 0 0 0 0 62.5 87.5 0 0 0 0 37.5 37.5 Monoplegia 0 0 0 0 0 75 0 0 0 0 0 0 Death 0 0 0 0 87.5 100 0 0 0 0 75 87.5 Table 1. Pathological manifestations of M3-infected, M5-infected, or mock-infected mice infected with WT virus (%; n = 8).
Further dynamic detection of immunity after viral challenge suggested that the immune response induced by M5 presented an increased anti-HSV-1 neutralizing antibody titer of 1:128 (Fig. 5A, 5B) and high levels of IFN-γ-specific lymphocyte proliferation from days 8 to 12 post challenge, which was clearly higher than that in the groups infected with the M3 or WT strains (Fig. 5C, 5D). Moreover, the viral loads in CNS tissues, especially various parts of the cerebrum and trigeminal ganglion in M3- or M5-infected mice, suggested a lower trend than those detected in tissues from the mock-infected mice (Fig. 5E–5H). Importantly, the tissue samples from the challenged individuals presented pathologic characteristics similar to those of tissues from animals infected with only M5. However, the CNS tissues of mock-McK and mock-17+ mice showed an obvious inflammatory reaction with extensive inflammatory cell infiltration, including aggregation of these cells around vesicles and hyperemia in the cerebrum (Fig. 5I). All these results indicated that M5 is capable of eliciting immunity against WT virus infection.
Figure 5. The immunity induced by M3 or M5 is capable of inhibiting viral replication in infected individuals. A, B The neutralizing capability of antibodies specific for HSV-1 in M3-, M5-, or mockinfected and HSV-1 WT-challenged mice. The geometric mean titers (GMTs) of neutralizing antibodies were measured by a neutralization test as described in the methods. C, D ELISpot responses to IFN-γ-secreting cells from splenic lymphocytes in M3-, M5-, or mockinfected and HSV-1 WT-challenged mice. Splenic lymphocytes were incubated for 24 h in the presence of a stimulus. The positive control was PHA. E, G Viral load in the brain (E) or trigeminal ganglion (G) of M3-, M5-, or mock-infected and WT-challenged mice at 28 days post infection (d.p.i.). F, H Viral load in the brain (F) or trigeminal ganglion (H) of M3-, M5-, or mock-infected and WT-challenged mice at 56 d.p.i. The values are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. I Pathological changes in the cerebral tissues of mice challenged with HSV-1 WT virus (4, 8, or 12 d.p.i.) at 28 or 56 d.p.i. Tissue sections were stained with H&E and imaged using an optical microscope, scale bars = 100 μm. Tissue hyperemia and infiltration of inflammatory cells are indicated with red arrows.
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The above results indicated that the effective immunity induced in M5-infected mice, which presented as humoral and cellular responses, was capable of controlling the viral proliferative capacity of the M5 strain and the WT strain in various organs of mice. Therefore, the analysis of latent HSV-1 infection in the trigeminal ganglion was a key point for evaluating the immunity induced by the M5 strain with deficient inhibition of cellular apoptosis. Using in situ hybridization with a primer specific for the LAT gene, we detected the level of LAT transcripts in trigeminal ganglion tissues from M3- or M5-infected mice subjected to subsequent WT challenge and mock-McK or mock-17+ -infected mice. The results showed a stronger hybridized transcript signal in the trigeminal ganglion of mock-McK and mock- 17+ mice at 12 d.p.i., and a weaker or absent LAT transcript signal was observed in the M3- or M5-infected mice (Fig. 6A). To verify this observation, trigeminal ganglion tissue samples were homogenized and co-cultured with a monolayer of Vero cells to observe cytopathic effects. The results showed a significant cytopathic effect in the co-culture system with samples from mock-McK- and mock17? -infected mice after only 5 days, but the Vero cells co-cultured with the samples from M3- or M5-infected and challenged mice remained negative after 12 days (Fig. 6B). This finding suggested that latent viral infection in the trigeminal ganglion was inhibited in M5-infected and challenged mice.
Figure 6. Latent infection of HSV-1 WT is reduced in mice infected with M3 or M5. A The levels of LAT expression in the trigeminal nerves of mice at 12 days post challenge with WT virus after 28 or 56 d.p.i., as determined by in situ hybridization. Positive LAT RNA expression in the trigeminal nerves of mice by chromogenic in situ hybridization is indicated with red arrows. Brown dot positivity is observed in mock-infected mice. No positive signal was observed in M3- or M5-infected mice, scale bars = 100 μm. B Vero cells cocultured with trigeminal ganglion tissues from M3-, M5-, or mockinfected and WT-challenged mice at 12 days post viral challenge. CPE is indicated by red arrows; scale bars = 100 μm.