Variant-specific tropism of human herpesvirus 6 in human astrocytes

Abstract

Though first described as a lymphotropic virus, human herpesvirus 6 (HHV-6) is highly neuropathogenic. Two viral variants are known: HHV-6A and HHV-6B. Both variants can infect glial cells and have been differentially associated with central nervous system diseases, suggesting an HHV-6 variant-specific tropism for glial cell subtypes. We have performed infections with both viral variants in human progenitor-derived astrocytes (HPDA) and monitored infected cell cultures for cytopathic effect (CPE), intra- and extracellular viral DNA load, the presence of viral particles by electronic microscopy, mRNA transcription, and viral protein expression. HHV-6A established a productive infection with CPE, visible intracellular virions, and high virus DNA loads. HHV-6B-infected HPDA showed no morphological changes, intracellular viral particles, and decreasing intra- and extracellular viral DNA over time. After long-term passage, HHV-6B-infected HPDA had stable but low levels of intracellular viral DNA load with no detectable viral mRNA. Our results demonstrate that HHV-6A and HHV-6B have differential tropisms and patterns of infection for HPDA in vitro, where HHV-6A results in a productive lytic infection. In contrast, HHV-6B was associated with a nonproductive infection. These findings suggest that HHV-6 variants might be responsible for specific infection patterns in glial cells in vivo. Astrocytes may be an important reservoir for this virus in which differential tropism of HHV-6A and HHV-6B may be associated with different disease outcomes.

FIG. 1.

Detection of HHV-6 gp116/54/64 glycoproteins in human progenitor-derived astrocytes and in T lymphocytes. (A) SupT-1 cells (left images) infected with either HHV-6A (U1102) or with HHV-6B (Z29) show bright expression of viral glycoproteins gp116/54/64 (green) in the cytosol. In contrast, mock-infected HPDA (right image) stain positively with an antibody against GFAP (green) and show no unspecific staining for HHV-6 glycoproteins (lack of red signal). (B) Double IFA for viral glycoproteins (red, arrows) and GFAP (green) on HPDA at 3 days postinfection with HHV-6. HPDA form syncytia following infection with the HHV-6A strains U1102 or GS but not with HHV-6B strain Z29. Magnifications, ×20 (A), ×32 (B, top and center panels), and ×20 (B, bottom panels).

FIG. 2.

Detection of viral particles in HHV-6A-infected HPDA by electron microscopy. Viral particles (thin arrows) are present in the nucleus (A) as well as in the cytosol at different stages of maturation (B) of infected HPDA, identified by characteristic glial filaments (thick arrow), at day 7 after infection. Viral particles are also visible extracellularly (B). Magnification, ×17,000.

FIG. 3.

Quantitative detection of intracellular (filled circles) and extracellular (empty diamonds) HHV-6 DNA at 3 h and 1, 3, 5, and 7 days postinfection of HPDA and SupT-1 cells. While both viral variants (U1102 and Z29) establish a productive infection in SupT-1 cells, only infection with U1102 is productive in HPDA. Intracellular DNA is expressed as viral copies/10⁶ cells; extracellular DNA is expressed as viral copies/milliliter ± standard error of the mean of triplicate values. I, inoculum.

FIG. 4.

Quantitative detection of intracellular viral DNA in HHV-6B-infected HPDA at subsequent passages and 3 days after PMA treatment. In these cultures, the viral load decreases with number of passages; treatment of infected cultures with PMA has no effect on the viral load. Each bar with the same filling represents one separate experiment.

FIG. 5.

Detection of viral transcripts by RT-PCR in HPDA infected with HHV-6A or HHV-6B. RT-PCR for U33, U12, U39, U27, and U94 at day 3 postinfection is shown. Each set of primers was tested on HPDA cDNA (S), nonretrotranscribed RNA as a negative control (C−), and a positive control of cDNA obtained from SupT-1 cultures infected with either viral variant (C+). (A) HPDA infected with U1102. (B) HPDA at day 3 postinfection with HHV-6B strain Z29. (C) After the third passage, no viral transcripts were detected in HPDA infected with Z29. β-actin was used as an internal control.

FIG. 6.

Viral load in HPDA 7 days after infection with HHV-6A strain U1102 and effect of blocking monoclonal antibodies against CD46. Preincubation of HPDA with the monoclonal antibody M177 (against SCR2), but not with M160 (against SCR3), J4.48 (against SCR1), or antibody anti-HLA-DR, significantly decreases the viral load in these cells compared to control (no antibody).