Figure 1.
Deep sequencing analyses of HERVs in healthy brain.
(A) Deep sequencing of the fetal and surgically resected (Surg) brain samples revealed that HERV-H exhibited the highest tag frequency and median number of tags followed by HERV-K. (B) When analyzing the HERV-K tags, LTR tags were most abundant, followed by gag-pol and then the env region tags (tags were normalized to respective gene lengths) (C) All host genes with transcript expression profiles correlated with HERV-K(II) env tag abundance (r2≥0.5) were analyzed using the DAVID tools [58] for enriched gene ontology (GO) terms. Genes related to cell cycle functions and chromosomal organization were most strongly associated with HERV-K(II) env expression. With the use of DAVID bioinformatics resources [59], the predicted target genes were classified according to KEGG functional annotations to identify pathways that were actively regulated by HERV-K(II) env transcripts in brain tissue. The most over-represented GO term belonged to the transcriptional regulation and chromosome organization followed by different stages of cell cycle pathway. (Mann Whitney t test, *p<0.05, **p<0.01).
Figure 2.
Activation of HERV-K(II) env by cAMP and EGF in different human cell lines
(A) Individual cell lines displayed differential constitutive HERV-K(II) env expression profiles. (B) Upon treatment of human fetal neurons, db-cAMP did not have any effects on HERV-K(II) env expression but EGF down-regulated HERV-K(II) expression. (C) U937 and (D) HFA showed decreased in HERV-K(II) env expression upon both db-cAMP and EGF exposure. (n = 4 replicates per group across two independent experiments).
Figure 3.
Over expression of HERV-K(II) Env exerts neurotrophic effects:
(A) Transfection of the pHERV-Kenv plasmid into SK-N-SH cells showed HERV-K(II) Env immunoreactivity at the predicted molecular weight on western blot. (B) Upon treatment with supernatants from SK-N-SH cells transfected with pHERV-Kenv plasmid, HFN showed increases in BDNF and NGF transcript abundance compared to the control vector transfected cells. (n = 3, with technical quadruplicates) (C) βIII-tubulin expression in HFN following 24-hour exposure to supernatants from HFA-transfected with the pHERV-Kenv or the control vector, showing an increase in βIII-tubulin immunoreactivity in cells exposed to HERV-K Env-transfected cells. (n = 2, with technical octuplicates) (Student t test, *p<0.05, **p<0.01).
Figure 4.
HERV transcripts in HIV− infected brain specimens.
(A) Deep sequencing of the HIV− and HIV+ autopsied cerebral white matter revealed a higher tag frequency of HERV-K in both clinical groups compared to other HERVs. (B) With the use of the DAVID bioinformatics resources, the predicted target genes were classified according to KEGG functional annotations to identify pathways that were actively regulated by HERV-K(II) env transcripts in brain tissue.
Figure 5.
Brain expression of HERV-K(II) Env in HIV/AIDS:
(A) HERV-K(II) env transcript analysis of HIV−) and HIV+ brains revealed high levels of HERV-K(II) env in cortex of HIV+ as compared to HIV− brains. (B) Immunohistochemical analyses of brain sections from HIV+ patients showed increased immunoreactivity of HERV-K(II) Env (arrow) protein in neurons as compared to the HIV− brain sections. HERV-K(II) Env protein expression co-localized in neurons expressing MAP-2 (insert: brown, MAP-2; blue, HERV-K Env). (C) In cerebral cortical specimens, HIV+ patients exhibited higher levels of HERV-K(II) Env detection than HIV− patients on immunoblotting of HERV-K(II) Env protein. (D) Quantitation of HERV-K(II) Env/β-actin band density on immunoblots (Original magnification: B-400X; insert, 200X). (Student t test, **p<0.01).
Figure 6.
HERV-K(II) env transfection of neuronal cells was neuroprotective.
(A) Analyses of SK-N-SH cells transfected with the pHERV-Kenv plasmid compared to the control (pGFP) showed that the efficiency of transfection was ∼20% (n = 3, with technical triplicates). (B) HERV-K(II) Env immunoreactivity was minimally detected in cells transfected with the control vector. pHERV-Kenv-transfected cells showed HERV-K(II) Env immunoreactivity at low (C) and high magnification (D). (E) Comparison of BDNF and NGF transcript levels in SKN-N-SH cells transfected with pGFP or pHERV-Kenv. (F) Exposure of pHERV-Kenv and control vector-transfected NG108 cells to staurosporine, HIV-1 Vpr or NMDA, showed that pHERV-Kenv-transfected cells were differentially protected depending on the neurotoxin. (Student t test, *p<0.05, ***p<0.001).
Figure 7.
Neural stem cells expressing HERV-K(II) Env are protective in vpr/RAG1−/− animals.
(A) Schematic of representation of C17.2 implantation site (marked by the •) in Vpr/RAG1−/− mice. (B) Western blot showing HERV-K(II) Env immunoreactivity in transfected cells. (C) TNF-α expression was suppressed in the brains of animals implanted with cells expressing HERV-K(II) env while (C) IL-6 was induced. Nissl staining showed similar striatal neuronal densities in animals implanted with cells transfected with either pGFP or pHERV-Kenv/pGFP (E, I). Immunohistochemistry revealed lower expression the microglia protein, Iba-1 (K) and higher expression levels astrocyte protein, GFAP (L) in HERV-K(II) env implanted brains compared to control vector (pGFP) implanted animals (F, G), respectively. Cleaved caspase-6 immunoreactvity was comparative reduced in striatum of animals receiving cells transfected with pHERV-Kenv/pGFP (M) but BDNF immunoreactivity was increased in the same animals (N) compared to controls (H, I). (O) At days 7 and 14, neurobehavioral deficits were greater in terms of ipsiversive rotations among the animals implanted with c17.2 cells transfected with the pGFP vector. (Original magnification: E–J, 400X) (Mann-Whitney test, *p<0.05).
Table 1.
Oligonucleotide primers used in Real-time RT PCR analyses.