Infection with an endemic human herpesvirus disrupts critical glial precursor cell properties

Abstract

Human herpesvirus 6 (HHV-6), a common resident virus of the human CNS, has been implicated in both acute and chronic inflammatory--demyelinating diseases. Although HHV-6 persists within the human CNS and has been described to infect mature oligodendrocytes, nothing is known about the susceptibility of glial precursors, the ancestors of myelin-producing oligodendrocytes, to viral infection. We show that HHV-6 infects human glial precursor cells in vitro. Active infection was demonstrated by both electron microscopy and expression of viral gene transcripts and proteins, with subsequent formation of cell syncytia. Infection leads to alterations in cell morphology and impairment of cell replication but not increased cell death. Infected cells showed decreased proliferation as measured by bromodeoxyuridine uptake, which was confirmed by blunting of the cell growth rate of infected cells compared with uninfected controls over time. The detailed analysis using novel, fluorescent-labeled HHV-6A or HHV-6B reagents demonstrated strong G1/S phase inhibition in infected precursor cells. Cell cycle arrest in HHV-6-infected cells was associated with a profound decrease in the expression of the glial progenitor cell marker A2B5 and a corresponding increase in the oligodendrocyte differentiation marker GalC. These data demonstrate for the first time that infection of primary human glial precursor cells with a neurologically relevant human herpesvirus causes profound alterations of critical precursor cell properties. In light of recent observations that repair of CNS demyelination is dependent on the generation of mature oligodendrocytes from the glial precursor cell pool, these findings may have broad implications for both the ineffective repair seen in demyelinating diseases and the disruption of normal glial maturation.

Figure 1.

Human glial precursor cells express the surface receptor CD46 and can be infected with HHV-6. Glial precursors were adhered overnight to fibronectin/laminin-coated coverslips before being fixed and immunostained with antibodies to CD46 (red) and counterstained with DAPI (blue) to reveal cell nuclei (A). Approximately 70–80% of cells demonstrated expression of CD46. Glial precursors were infected with HHV-6A (B) or HHV-6B (C), stained with antibodies to the HHV-6 p41 and p101 proteins (green), and counterstained with DAPI to highlight cell nuclei (blue). Approximately 10–20% of cells exposed to either virus strain expressed viral proteins. No viral protein expression could be detected in unexposed precursor cells (D). Scale bars, 15 μm.

Figure 2.

HHV-6-infected and uninfected control precursor cells express IE (U86) and late (U47) gene transcripts as determined by RT-PCR. Lanes 3 and 4 show a 370 bp HHV-6 amplicon from the U86 gene. Lanes 6 and 7 show amplicons from gene U47 of HHV-6 strains A and B, respectively. Note that the U47 A gene contains a 150 bp deletion with respect to the U47 B gene within the region defined by the primers. No amplicons are generated from uninfected precursor cells as shown in lanes 5 and 8.

Figure 3.

HHV-6B-infected human glial precursor cells show morphological changes as determined by light and electron microscopy. Cells were grown on fibonectin/laminin-coated coverslips for 5 d before being fixed and stained with antibodies against HHV-6 gp 116 (green) and counterstained with DAPI (blue). Uninfected cells demonstrated characteristic polygonal cell morphology with relatively few processes characteristic of normal human glial precursor cells (A). In contrast, HHV-6B-infected cells examined at the same 5 d time point exhibited morphological changes characterized by development of increased numbers of radial processes accompanied by multinucleated syncytia (B). Morphological changes in HHV-6B-infected precursor cells examined by electron microscopy show extensive vacuolization and contain nests of dense core particles not seen in controls (magnification, 4000×) (C). Later in infection with HHV-6B (or HHV6-A; data not shown), glial precursors formed multinucleated syncytia (magnification, 1500×) (D). A high-power image of an HHV-6B-infected vacuole (magnification, 40,000×) shows virions consistent in size and morphology with HHV-6 (E); spherical intracellular viral particles possessing an electron-dense core, tegument, and capsid were identified (arrow). No vacuolization or cell syncytia were seen in uninfected glial precursors (magnification, 2500×) (F). Scale bar (in A): A, B, 15 μm.

Figure 4.

HHV-6-infected glial precursor cells showed a decrease in BrdU incorporation that was accompanied by reduced cell growth in culture over time. A, Precursor cells show decreased BrdU incorporation after HHV-6 infection. Quantification of BrdU-positive glial precursor cells was expressed as a percentage of total cells. Precursor cells were either exposed or sham exposed to HHV-6 overnight, fluorescence-activated cell sorted, and incubated for 48 hr before being pulsed with BrdU for the final 8 hr. Proliferation of precursor cells was significantly reduced by infection with both the A and B strains of virus. p values are expressed relative to uninfected control precursor cells (*p < 0.0016; **p < 0.0013). B, Glial precursor cells infected with HHV-6A or HHV-6B showed markedly diminished cell numbers over time compared with uninfected controls. Cells were infected with HHV-6A or HHV-6B overnight before being fluorescence-activated cell sorted in parallel with uninfected controls and plated at the same density on coverslips in optimal growth conditions in triplicate. Medium was changed every other day, cells were stained with DAPI on the day indicated, and unbiased counting of all cells was performed. Cell numbers of infected populations were ∼55–60% of controls by day 5 after infection.

Figure 5.

HHV-6A and HHV-6B infection of human glial precursor cells result in cell cycle arrest. Human glial precursors were infected with FITC–HHV-6 for 72 hr, fixed, and analyzed for DNA content by FACS. Uninfected controls show the characteristic profile of proliferating glial precursors, with the majority of cells in G₀ /G₁ (A, A′). The effects of HHV6-A (B) or HHV-6B (B′) infection on precursor cell cycling cannot be visually discriminated from each other or the uninfected controls. The G₂ /M blocking agent nocodazole caused uninfected precursors to accumulate in G₂ /M (C, C′). In D and E, as well as (D′) and (E′), precursors were exposed to FITC–HHV-6A and FITC–HHV-6B, respectively, followed by a terminal 24 hr incubation in nocodazole. HHV-6-negative (D, D′) or HHV-6-positive (E, E′) cells from the same flask were then analyzed for DNA content. The nocodazole treatment permits the discrimination of the G₁ /S block induced by prior infection with either HHV-6A or HHV-6B (E and E′, respectively) in comparison with uninfected controls (D and D′). F shows a summary of the results of HHV-6A and HHV-6B infection on glial precursor cell cycling from five independent experiments. The error bars indicate SEM.

Figure 6.

HHV-6-infection of human glial precursor cells was not associated with increased cell death. LDH assay of precursor cell supernatants demonstrates no significant differences in LDH leakage attributable to cell membrane damage between HHV-6-infected and uninfected control cells (A). TUNEL assay for apoptotic cell death also showed no significant difference between HHV-6-infected and uninfected control cells examined 5 d after infection (B).

Figure 7.

Infection with HHV-6A and -6B inhibits self-renewal and promotes differentiation of human glial progenitor cells. A representative fluorescence microscopic field from uninfected versus HHV-6B-infected precursor cells immunostained for GalC (red) and counterstained with DAPI (blue) for cell nuclei (A) is shown. Human glial progenitors were exposed overnight to either FITC-labeled HHV-6A or HHV-6B before being separated on a fluorescence-activated cell sorter and plated on coverslips. After a 48 hr incubation in a medium that allows for oligodendrocytic differentiation, cells were fixed and immunostained. The histogram (B) shows that a higher percentage of GalC⁺ cells was seen in the HHV-6-infected cell population. Uninfected precursor cell cultures (controls) contained ∼55–60% A2B5⁺ progenitor cells compared with only 25–30% in either HHV-6A- or HHV-6B-infected cells. Conversely, in the HHV-6A- or HHV-6B-infected populations, ∼70% of cells expressed the oligodendrocyte differentiation antigen GalC, compared with 40–45% in the uninfected controls. The percentage of cells coexpressing both A2B5 and GalC was small and did not change significantly with infection. *p < 0.01; **p < 0.035). Scale bars, 75 μm.