Expression of the Human Herpesvirus 6A Latency-Associated Transcript U94A Disrupts Human Oligodendrocyte Progenitor Migration

Abstract

Progression of demyelinating diseases is caused by an imbalance of two opposing processes: persistent destruction of myelin and myelin repair by differentiating oligodendrocyte progenitor cells (OPCs). Repair that cannot keep pace with destruction results in progressive loss of myelin. Viral infections have long been suspected to be involved in these processes but their specific role remains elusive. Here we describe a novel mechanism by which HHV-6A, a member of the human herpesvirus family, may contribute to inadequate myelin repair after injury.

Figure 1

CD140+ hOPCs express common OPC markers and differentiate into oligodendrocytes and astrocytes in vitro. hOPCs were isolated from 19–22 week fetal human tissue by immunomagnetic purification using anti-CD140 beads. When cultured in the presence of PDGF and FGF for 7 days, these cells expressed a variety of known OPC markers, including (a) PDGFRα, (b) Nkx2.2, (c) CNPase, (d) A2B5, (e) NG2, and (f) PSA-NCAM. (g) When exposed to bone morphogenic protein 4 (BMP-4) for 7 days, cells differentiated into GFAP+ astrocytes. (h) When exposed to triiodothyronine/thyroxine (T₃/T₄) for 10 days, these cells differentiated into GalC+ oligodendrocytes.

Figure 2

U94A expression inhibits hOPC migration in vitro. (a) Nested rtPCR shows that hOPCs infected with HHV-6A express U94A mRNA at 4 and 10 days post-infection. (b) Lentiviral expression of U94A RFP significantly inhibits hOPC migration as analyzed by an agarose drop assay. (c) U94A RFP expression does not affect hOPC proliferation over the course of 21 days in vitro. (d) Proportion of dividing hOPCs, determined by BrdU incorporation after 4, 24, and 48 hours of incubation. (e,f) U94A RFP expression does not significantly affect OPC differentiation into GFAP+ astrocytes or GalC+ oligodendrocytes in vitro. Data from 3–6 independent experiments, normalized to GFP+ controls, pooled and displayed as mean ± standard error of the mean (SEM); ns = not significant; ***p < 0.001 versus control. Effects of U94A expression on hOPC proliferation and BrdU incorporation were analyzed by two-way ANOVA. Effects of U94A expression on hOPC migration and differentiation were analyzed by unpaired student’s t-test. Nested rtPCR agarose gel shown as cropped image. Full images shown in supplementary information.

Figure 3

U94A expression inhibits hOPC migration in vivo but does not prevent oligodendrocyte differentiation. (a) hOPCs expressing U94A (RFP) or controls (GFP) were injected bilaterally into the corpus callosum and hippocampus of cuprizone-treated NSG mice. Brains were analyzed 21 days after transplantation. Representative confocal images from (b) corpus callosum white matter and (c) hippocampus. (d) U94A RFP+ hOPCs migrated significantly shorter distances than GFP+ controls. Cell dispersion was measured using stereological measurements of the distance of each cell to the center point of the transplanted cell population. Measurements averaged from at least three different sections from each of four different animals. (e) Analysis of NSG brains at 21 days post- transplant show that a majority of GFP+ and U94A RFP+ hOPCs differentiated into GSTπ positive oligodendrocytes (arrowheads). (f) No GFP+ or U94A RFP+ hOPCs were observed to differentiate into GFAP+ astrocytes (asterisks). Cell dispersion data displayed as mean ± SEM; *p < 0.05 versus GFP+ control. Effect of U94A expression on hOPC cell dispersion was analyzed by unpaired student’s t-test.