Fig 1.
Characterization of herpes simplex virus 1 (HSV-1) genomes during establishment of latency.
(A) DNA-FISH detection of HSV-1 genomes (red). (i) HSV-1 replication compartment (RC) pattern (ii) HSV-1 multiple-acute (MA) pattern. Black/white middle images represent staining of the cellular DNA with DAPI. (B) The HSV-1 genome patterns detected during establishment of latency (from 4 to 28 dpi) are presented as colored and black-and-white DNA-FISH images (up), and drawings (down). Patterns detected were: RC; MA; multiple-latency (ML); four, three, two spots (4-3-2); and single (S) or single+ (S+). The relative proportions of each pattern are signified by “+” or “–“.“-“: pattern was not observed; “+”: less than 10% of the observed patterns; “++”: between 10% and 25% of the patterns; and “+++”: more than 25% of the patterns. Dashed lines indicate nuclei. (C) Immuno-DNA-FISH showing HSV-1 genomes (red, 6 dpi), neurofilament (NF160) protein (green), and cellular chromatin (DAPI, blue). Nu: nucleus; Cy: cytoplasm. (D) Immuno-DNA-FISH showing HSV-1 genomes (red, 6 dpi), viral proteins (green), and cellular chromatin (DAPI, blue/grey). (E) RNA-DNA FISH showing HSV-1 genomes (red) and LAT (blue/grey). Inset at top right in (vi) shows LAT expression; inset at bottom right in (vi) shows the same image overexposed to visualize the small viral dots (arrows) present in the nucleoplasm. RC: Replication Compartment; MA: multiple-acute; ML: multiple-latency; S: single; S+: single +. Scale bars represent 10 μm.
Fig 2.
HSV-1 MA pattern corresponds to vDCP-NBs and contains SUMO proteins.
(A) Immuno-DNA-FISH showing HSV-1 genomes (red), promyelocytic leukemia nuclear bodies (PML-NB)–associated proteins (green), and cellular chromatin (DAPI, blue). (B) WB of ATRX and Daxx in TG of uninfected or infected mice during acute infection. Infected (left TG) and not-infected (right TG) TGs of the same mouse (two mice) were harvested 6 dpi, and treated to perform WB. Actin is shown as a loading control. NIH3T3 is shown as a cellular control. (C) and (D) Immuno-DNA-FISH showing HSV-1 genomes (red), small ubiquitin modifier (SUMO) proteins (green), and cellular chromatin (DAPI, blue/grey). (C) SUMO-1 and (D) SUMO-2/3 detection in (i) non-infected neurons, (ii) RC-containing neurons and (iii, iv) MA/vDCP-NBs or S/vDCP-NB-containing neurons. (E) IF for detection of PML (green) in uninfected TG neurons and satellite cells. Arrows point out PML-NBs in neurons or satellite cells, arrowheads point out neurons without PML-NBs. (F) Immuno-DNA-FISH showing HSV-1 genomes (red, RC) and (i) PML or (ii) CENP-A (green), in neurons. DAPI staining (grey) shows nuclei. Scale bars represent 10 μm.
Fig 3.
Behavior of PML-NB-associated proteins in HSV-1 infected cultured primary mouse TG neurons.
(A) Immuno-FISH detection of neuronal or viral markers (green) and HSV-1 genomes (red) in RC-containing neurons. (i) neurofilaments, (ii) ICP4, (iii) ICP27, (iv) total HSV-1 proteins. (B) Immuno-FISH detection of PML-NB components (green) and HSV-1 genomes (red) in RC-containing neurons. (C) Quantification of infected neurons showing co-localization of HSV-1 genomes with PML-NB components. Fifty to 80 neurons were counted for each labeling. (D) Quantification of RC-containing infected neurons showing or not PML aggregates. Scale bars represent 10 μm.
Fig 4.
vDCP-NBs are formed in the absence of functional ICP4 and ICP0 in cultured primary mouse TG neurons, and can be associated with viral transcription.
(A) Neurons were infected with in1374 at 32°C for 24 h. (i) IF detection of ICP4, (ii and iii) immuno-FISH for detection of ICP4 or ICP8 and HSV-1 genome, and (iv–vii) immuno-FISH for the detection of PML, Daxx, ATRX or SUMO-2/3 and the HSV-1 genome. (B) and (C) Neurons were infected at 38.5°C for 48 h with in1374 and in1330, respectively. (i–iv) immuno-FISH for the detection of PML, Daxx, ATRX or SUMO-1 and the HSV-1 genome. (D) RT-qPCR for the detection of LacZ transcripts in in1374 infected neurons treated or not with trichostatin A (TSA). Results show means (± SD) of two independent experiments. (E) RNA-DNA FISH combined with IF for detection of LacZ transcripts (blue), HSV-1 genomes (red), and PML (green) in in1374 infected neurons treated or not with TSA. Three different patterns are shown. (F) RNA-DNA FISH combined with IF for detection of LAT transcripts (blue), HSV-1 genomes (red), and PML (green) in mouse TG neurons at 6 to 8 dpi. Scale bars represent 10 μm.
Fig 5.
Type 1 IFNα induces an ML-like pattern in infected cultured primary mouse TG neurons.
(A) Immuno-FISH for the detection of PML and HSV-1 genomes in cells not treated (i) or treated (ii) with IFNα (1000 IU/mL). (B) Double DNA FISH for the detection of centromeric minor satellite sequences and HSV-1 genomes in cells treated with IFNα (1000 IU/mL). (C) Quantification of ML-like and RC patterns in neurons (three independent experiments) infected with in1374, in1330, or tsK for 24 h in the absence or presence of IFNα (1000 IU/mL). Results show means (± SD). (D) Quantification of ML-like and RC patterns in neurons from C57BL/6 wt or IFNAR KO mice, and infected by in1374 for 24 h in the absence or presence of IFNα (1000 IU/mL). Means of two independent experiments are shown.
Fig 6.
Detection of HSV-1 genomes in neurons from human TGs.
(A) Table showing the characteristics of the five patients from which TGs were analyzed. (B) PCR for the detection of HSV-1 genomes in the five TGs harvested from patient 1 to 5. TK and LAT loci were detected. The lane HSV-1 indicates viral genomes detection in infected cells used as a positive control. C is the negative control issued from uninfected cells. (C) RT-PCR for detection of several lytic transcripts and LAT in the five human TGs. C: RNA from controlled infected cells (for ICP0, 4, 27, UL30 and VP16 detection) or mouse TG (for LAT detection). RT is for Reverse Transcription. “-”and “+” indicate samples without or with reverse transcription, respectively. (D) IF for the detection of three neurofilament markers (NF160, NF200 and ßIII-tub). (E) IF for the detection of PML and neurofilaments. (F) RNA FISH for LAT detection using tyramide 488 (i, green) or tyramide 350 (ii, blue). (G) RNA-DNA FISH combined with IF for the detection of LAT transcripts (blue), HSV-1 genomes (red), and PML (green). Three different PML patterns are shown. The PML signal in non-neuronal cells is shown in (i). Scale bars represent 10 μm.