Critical role for a high-affinity chemokine-binding protein in γ-herpesvirus–induced lethal meningitis (original) (raw)
Targeted disruption of the M3 open reading frame. We disrupted the M3 open reading frame (ORF) by inserting a translational stop with an accompanying frameshift mutation into the _Acc_I site at genomic coordinate 7176 (18) (γHV68-M3.stop virus; Figure 1a). This site is 34 amino acids into the 406 amino acids of the ORF, but is downstream from the end of the secretion signal peptide, such that truncated versions of the protein potentially made by downstream translation initiation should not be secreted from the cell.
Construction and verification of the γHV68-M3.stop and γHV68-M3.MR viruses. (a) Genomic structure of γHV68, γHV68-M3.stop, and γHV68-M3.MR in the region of the M3 ORF. (b) Southern blot analysis of γHV68, γHV68-M3.stop, and γHV68-M3.MR. Viral DNA was purified from viral stocks, digested with _Avr_II, and analyzed with a probe spanning the M3 ORF (see a). The sizes of the hybridizing bands are indicated. (c) Western blot analysis of γHV68, γHV68-M3.stop, and γHV68-M3.MR. Total cellular lysate was analyzed with either a polyclonal Ab to the M3 protein or an mAb to β-actin. (d) Real-time RT-PCR analysis of M2 and M4 genes in NIH 3T12 cells lytically infected with either wild-type γHV68, γHV68-M3.stop, or γHV68-M3.MR. Gene 6 transcription was analyzed in parallel as a control. Shown are mean ± SEM of pooled data from at least three independent experiments. NS, no statistically significant difference between transcript levels in cells infected with γHV68-M3.stop compared with cells infected with either γHV68 or γHV68-M3.MR.
Purification of γHV68-M3.stop provided a homogenous stock, as determined by the presence of the inserted stop sequence in 50 out of 50 plaques (as determined by PCR analysis), absence of detectable M3 protein in 50 out of 50 plaques (as determined by ELISA), and appropriate digestion patterns on Southern analysis of 20 out of 20 plaques (data not shown). Southern analysis of γHV68-M3.stop by restriction digest with _Avr_II, using a 32P-labeled M3 region probe (bp 5362–7893), demonstrated, as predicted, the addition of an additional _Avr_II site within the M3 ORF (Figure 1b). Western analysis of NIH 3T12 cells infected with the γHV68-M3.stop virus indicated that no full-length M3 protein, or truncated products (data not shown), could be detected with a polyclonal Ab to the M3 protein (Figure 1c).
The M3 stop mutation lies 1,233 bp from the predicted translational start of the adjacent M4 gene and 2,548 bp from the known transcriptional start site of the adjacent M2 gene, making it unlikely that insertion of the M3 mutation would alter transcription of either M2 or M4 (24, 25). To determine if the M3.stop mutation altered M2 or M4 transcription, we performed real-time RT-PCR analysis of transcript levels in NIH 3T12 fibroblasts infected with wild-type γHV688, γHV68-M3.stop, or γHV68-M3.MR. Transcript levels from the M2 and M4 genes were not significantly altered by the presence of the M3.stop mutation (Figure 1d).
To rule out the presence of distal mutations in γHV68-M3.stop that might result in phenotypic alterations in γHV68-M3.stop, we constructed and purified a marker rescue virus (γHV68-M3.MR), which reconstituted wild-type M3 ORF sequence into γHV68-M3.stop. Southern analysis of γHV68-M3.MR after _Avr_II digestion demonstrated, as expected, the loss of the engineered _Avr_II site (Figure 1b), and Western analysis indicated expression of the M3 protein (Figure 1c).
M3 is not required for efficient replication in cultured cells, spleen, or lung. Based on our ability to isolate the γHV68-M3.stop mutant, the M3 ORF is nonessential for in vitro replication. To evaluate whether M3 has a role in in vitro replication, we compared γHV68-M3.stop and wild-type γHV68 during multiple rounds of replication in NIH 3T12 cells (Figure 2a). We found that M3 is not required for efficient replication in immortalized murine fibroblasts. Similarly, M3 was not required for efficient replication of γHV68 in either the spleen or lung of C57BL/6 (B6) mice 4 or 9 days after either intraperitoneal or intranasal inoculation (Figure 2b).
Replication of the γHV68-M3.stop virus. (a) Multistep growth curve. NIH 3T12 monolayers were infected with 0.05 PFU per cell of either γHV68 or γHV68-M3.stop, and samples were harvested at various times after infection. The mean and SEM of two independent experiments are shown. (b) C57BL/6 mice were infected with either 106 PFU intraperitoneally or 4 × 105 PFU intranasally and harvested at either 4 or 9 days after infection. Lung and spleen were assayed for viral titer. The mean ± SEM of two independent experiments (ten mice total per condition) are shown. i.p., intraperitoneal; i.n., intranasal.
M3 is essential for efficient induction of meningitis and virulence after intracerebral inoculation. Chemokine expression has been demonstrated to increase after CNS infection with numerous viruses (26–29), and chemokines have been postulated to play an important role in CNS viral pathogenesis (reviewed in ref. 30). Considering that γHV68 is capable of infecting numerous cell types within the brain (31), we tested the hypothesis that the M3 protein has a role after intracerebral infection of 21-day-old immunocompetent CD1 mice. γHV68-M3.stop was 100-fold less virulent than wild-type γHV68 after intracerebral inoculation (Figure 3a). This attenuation was specific to the mutation in the M3 ORF, since γHV68-M3.MR virus was equal in virulence to γHV68. A decrease in virulence of the γHV68-M3.stop compared with γHV68-M3.MR after intracerebral inoculation was also demonstrated in B6 mice, assuring that the attenuated phenotype observed in CD1 mice is not strain dependent (P = 0.0263, sixty 21-day-old BL6 mice challenged with 1, 10, and 100 PFU of γHV68-M3.stop or γHV68-M3.MR).
Virulence of γHV68-M3.stop virus. (a) CD1 mice (21 days old) were inoculated intracerebrally with various doses of γHV68, γHV68-M3.stop, or γHV68-M3.MR, and followed for 2 weeks. Each data point represents the mean ± SEM of three independent experiments (30 mice total per dose, *P < 0.0001). (b) γIFNR–/– mice (n = number of mice across five separate experiments) were infected with 106 PFU of either γHV68 or γHV68-M3.stop and followed for 84 days.
We considered that M3 might be required for efficient replication in the brain despite the lack of a requirement for M3 in replication in the spleen and lung (above). We therefore determined the viral titer within the brain, lung, and spleen at various days after intracerebral inoculation with 100 PFU of either γHV68-M3.stop, γHV68, or γHV68-M3.MR. At the peak of viral replication in the brain (day 5; Figure 4a), there was approximately a tenfold decrease in viral titer in the brain that was specific to the mutation in the M3 ORF (P = 0.0019). This decrease was considerably smaller at day 7 after inoculation. Consistent with data after intraperitoneal or intranasal inoculation (Figure 2), after intracerebral inoculation there was no role for M3 in viral replication in lung or spleen (Figure 4, b and c).
Viral titer after intracerebral infection with γHV68-M3.stop virus. CD1 mice (21 days old) were inoculated intracerebrally with 100 PFU of either γHV68, γHV68-M3.stop, or γHV68-M3.MR. At various points after infection, viral titer in the brain (a), spleen (b), or lung (c) was determined. Each data point represents the mean ± SEM of two independent experiments (ten mice total per condition). *P = 0.0019.
To assess the location and extent of viral replication within the brain, as well as the nature of the inflammatory response to the infection, we performed histological examination of the brains of infected mice. Examination of sections at days 5 and 7 after infection indicated discontinuous areas of meningeal inflammation in mice infected with either γHV68 or γHV68-M3.stop. Meningeal inflammation was most prominent in the region of the cerebellum and basal forebrain. Examination of these slides with Ab specific to γHV68 antigen indicated that virus was present at the sites of inflammation in similar patterns for both γHV68 (Figure 5, a–c) and γHV68-M3.stop (Figure 5, d–f).
Meningeal localization of viral antigen after infection with γHV68 and γHV68-M3.stop. Immunohistochemical localization of viral antigen after intracerebral inoculation of 21-day-old CD1 mice with 100 PFU of either γHV68 (a–c) or γHV68-M3.stop (d–f) demonstrates that viral antigen is present in the meninges of mice infected with either virus. (a and d) Hematoxylin and eosin–stained sections of brain. (b and e) Sections at same magnification, stained with a polyclonal Ab to γHV68 and a Cy-3–conjugated secondary Ab (red). Slides were counterstained with Heochst dye, staining nuclei blue. (c and f) Higher magnification of boxed areas in b and e. Immunohistochemistry with preimmune serum as the primary Ab showed no reactivity with adjacent sections. Sections shown are representative examples of six mice, infected on two separate occasions. The scale bars represent 250 μm for a, b, d, and e , and 50 μm for c and f.
Strikingly, the nature of the inflammatory infiltrate was different after infection with γHV68 compared with γHV68-M3.stop. γHV68 and γHV68-M3.MR infection was associated with a marked preponderance of neutrophils in the meninges (Figure 6, a, c, d, and f). In contrast, the proportion of lymphocytes and macrophages was increased in the meninges of mice infected with γHV68-M3.stop (Figure 6, b and e). Differential counts conducted in a blinded manner confirmed that lack of M3 was associated with a considerable change in the inflammatory infiltrate (Figure 6g). Mutation of M3 led to a statistically significant increase in lymphocytes and macrophages (P = 0.0449 and P = 0.0004, respectively) and a decrease in neutrophils (P < 0.0001) relative to infection with γHV68-M3.MR.
The M3 protein alters the inflammatory response to γHV68. Hematoxylin and eosin–stained sagital brain sections from the basal forebrain of CD1 mice infected with γHV68 (a and d), γHV68-M3.stop (b and e), or γHV68-M3.MR (c and f) 5 days previously. The boxed areas in a–c are shown at higher magnification in d–f. The arrows in d and f indicate neutrophils (PMN). The arrow in e indicates a lymphocyte (L). (g) Differential counts of meningeal infiltrates of mice infected 5 days previously with the indicated viruses. Shown are mean ± SEM of pooled numbers from two independent investigators performing differential counts in a blinded manner on sections from four mice infected with γHV68, eight mice infected with γHV68-M3.MR, and eight mice infected with γHV68-M3.stop (*P = 0.0449, **P = 0.0004, and ***P < 0.0001). Scale bars, 100 μm. Mono/Blast, monocytes or lymphoblasts.
Chemokine expression is induced during γ HV68-induced meningitis. Since M3 has an important role in neurovirulence and is a high-affinity CBP, we wished to determine whether M3 ligands are expressed during CNS infection with γHV68. We therefore performed a RNase protection assay to determine whether chemokine expression increases in response to infection with γHV68. As shown in Figure 7, there was no detectable expression of any of the chemokines assayed in either naive mice or in mice 5 days after mock infection. In contrast, expression of CC chemokines RANTES, MIP-1β, and MCP-1, as well as the CXC chemokine IP-10, were detected 5 days after infection with either γHV68-M3.MR or γHV68-M3.stop (Figure 7). These data support the hypothesis that M3 may regulate CNS disease via its capacity to sequester inflammatory chemokines.
Enhanced chemokine mRNA expression after intracerebral inoculation with γHV68. RNA was harvested from brains of naive mice, mock-infected mice, or mice infected with 100 PFU of either γHV68-M3.MR or γHV68-M3.stop and subjected to RNase protection analysis. Twenty micrograms of total RNA were added per sample, except for the positive control mouse splenocyte RNA lane, which contained 2 μg RNA. Differences in loading between lanes were at most tenfold (e.g., compare L32 in Mock, lane 2, with γHV68-M3.MR, lane 1), while differences in expression of chemokines were in the range of 600-fold (e.g., compare RANTES in Mock, lane 2, with γHV68-M3.MR, lane 1). Band intensity was quantified using the STORM Phosphoimager software package (Molecular Dynamics, Piscataway, New Jersey, USA).
M3 does not play a role in regulating chronic inflammation in immunocompromised mice. Since M3 is important for regulating inflammatory responses during acute γHV68 infection, we considered the possibility that the M3 protein may regulate chronic inflammatory responses to γHV68 infection. We therefore examined the role of M3 in inflammatory aortitis and splenic atrophy induced by γHV68 in IFNγR–/– mice infected for weeks to months with either γHV68-M3.stop or γHV68 (21, 32). After infection with 2 × 106 PFU of either γHV68-M3.stop or γHV68, INFγR–/– mice died with similar kinetics and frequency (Figure 3b). Histological examination of aortic inflammation and splenic atrophy in these mice indicated that M3 plays no role in either the penetrance or severity of splenic or aortic lesions caused by either virus (data not shown).
M3 is not required for establishment or reactivation from latency. Since the region of the M3 ORF is transcribed in latently infected tissues (33–35) and a recent study indicated a γHV68 mutant virus in which a LacZ cassette was placed in the M3 gene has a marked reduction in establishment of latency (36), we examined whether γHV68-M3.stop was capable of establishing a latent infection in B6 mice after either intranasal or intraperitoneal inoculation. At either 2 weeks (Figure 8, a–d) or 7 weeks after infection (data not shown), splenocytes and peritoneal exudate cells were assessed for their ability to reactivate from latency in an ex vivo limiting dilution analysis (22, 23). We found that similar frequencies of reactivating cells were recovered after infection with either γHV68-M3.stop or γHV68, regardless of route of inoculation. To measure persistent replication in samples, replicate cell aliquots were mechanically disrupted. This procedure kills more than 99% of cells, but has at most a twofold effect on viral titer (22), thus allowing experimental distinction between reactivation from latency (which requires live cells) and persistent replication of virus in tissues. No significant persistent replication of either virus was detected under any of the experimental conditions (Figure 8, a–d).
γHV68-M3.stop establishes and reactivates from latency normally. Sixteen days after inoculation with either 106 PFU intraperitoneally (a, b, e, and f) or 4 × 105 PFU intranasally (c, d, g, and h), splenocytes (a, c, e, and g) and peritoneal exudate cells (b, d, f, and h) were harvested from B6 mice. Ex vivo reactivation (a, b, c, and d) was monitored by plating cells over an indicator monolayer and monitoring for viral cytopathic effect 21 days after plating (square symbols). Duplicate cell samples were mechanically disrupted to control for persistent viral replication (triangle symbols, denoted as disrupted in the figure). (e, f, g, and h) Percentage of cells harboring viral genome was assessed through a limiting dilution nested PCR assay. Data are pooled from three independent experiments of five mice per infectious condition (15 mice total per condition).
Using a nested PCR to detect γHV68 genome-bearing (15) cells, we found that at 2 weeks after infection, similar frequencies of cells harbored γHV68 genome in both splenocytes and peritoneal exudate cells after infection with either γHV68-M3.stop or γHV68 (Figure 8, e–h), demonstrating that the M3 protein is not required for establishment of or reactivation from latency.