STED microscopy can reliably detect the viral genome

To examine how STED and confocal microscopy differ, we labeled DNA probes designed towards the terminal regions of the viral genome (Fig. 1a) with either DIG (green) or Biotin (red) to generate two different colored probes to the same region of the viral genome to determine. The human primary fibroblast cells (BJ cells) were infected with the 17+ strain of HSV-1 at multiplicity of infection (MOI) of 0.1 or 5 PFU/cell for 6 h. Due to the heterogeneity of cells and variation in the number of incoming viruses in each cell, viral replication time varies from one cell to another, and as a result, progressing from small but distinct early replication compartments to large fused late replication compartments occupying most of the host nucleus took about 6 h post-infection. At a lower MOI (0.1 PFU/cell), we observed more smaller replication compartments, while at a high MOI of 5 PFU/cell infection, larger fused compartments were typically observed [14].

Confocal microscopy was developed to offer greater resolution than regular fluorescent microscopes by rejection of out-of-focus noise [34, 35]. Fig. 1b-d were captured with confocal microscopy to show BJ cells at early stage of replication. Fig. 1b, c were from red and green channels, respectively. Figure 1d is an overlay of Fig. 1b and c, while Fig. 1e stands for the analysis results of Fig. 1d. Correlation coefficient (Rr), also known as Pearson’s correlation coefficient, ranges from −1.0 to 1.0. 0 indicates no correlation between two signals and −1.0 represents complete negative correlation. Overlapping coefficient (R) represents the colocalization frequency of two selected signals [36]. The Rr and R of Fig. 1d are 0.733 and 69.1 %, respectively (Fig. 1e), suggesting a moderate correlation between the two probes.

STED microscopy results were shown in Fig. 1f-h. Figure 1f, g were from red and green channels, respectively, Fig. 1h is overlay of Fig. 1f and Fig. 1g. While Fig. 1i, j are details with enlargement of partial Fig. 1h, which are indicated by white rectangles. Figure 1k stands for the analysis results of Fig. 1h. Unlike confocal microscopy, there is a much better overlap between red and green signals from STED (Fig. 1h). The center sections of the two color signals overlapped tightly (Fig. 1i, j). The Rr of the two signals is 0.910, and R is 90.2 % (Fig. 1k). Values are much higher than that from confocal results. The visual colocalization and the high values of Rr and R from STED analysis demonstrate that STED is able to detect viral genomes.

To determine how these probes behave at the late stage of viral replication compartments development, when individual replication compartments merges into large ones occupying most of the host nucleus, we infected BJ cells at a high MOI of 5 PFU/cell for 6 h and examined the signals by confocal (Fig. 1l-n) and STED microscopy (Fig. 1p-r). Figure 1l (red signal), 1 M (green signal) are merged in Fig. 1n and related parameters are shown in Fig. 1o. Though the Rr and R of confocal image Fig. 1n are 0.650 and 77.5 % (Fig. 1o), respectively, there is still no macroscopic overlapping between two signals under the confocal microscopy, indicating that confocal microscopy again failed to convincingly colocalize the two signals.

In contrast, Fig. 1p (red probe) and 1Q (green probe) exhibite stronger correlations when merged in Fig. 1r and analyzed in Fig. 1u. Figure 1s, t are details with enlargement of partial Fig. 1r (white rectangles) to show overlapping red and green signals. In Fig. 1s, two color signals overlapped completely, and in Fig. 1t, just part of the signals overlapped. Under the STED microscopy, about 76.1 % of the two color signals overlapped (Fig. 1r). The Rr of Fig. 1r is 0.637 (Fig. 1u). Comparing Fig. 1h and Fig. 1r, both Rr and R decrease with the development of replication compartments.

As each DNA strand of the viral genome stochastically hybridize to red or green probes, the chances of a perfect overlap between red and green signals is approximately 25 % when there is abundant amount of probes present, such as at early stage of replication compartments development. In cells where viral replication compartments are well developed, there are a larger number of viral genomes, and a limited amount of probes present, which would result in an increased possibility of only one colored probe hybridizing to a single viral genome, thus the observed reduction of overlapping signals, and hence the decrease in Rr and R from STED imaging. The lack of changes in the Rr and R values from confocal imaging suggests that the confocal microscopy is intrinsically unreliable to describe the details needed for HSV-1 genomes.

Replication renders compact HSV-1 genomes into relaxed structures

When HSV-1 DNA enters the host nucleus, it assumes a condensed structure, with a diameter of 35–40 nm and a length of 130–160 nm [37]. The interaction between HSV-1 genome and host core histones occurs as early as 1 h post-infection, and the viral genome forms a nucleosome-like structure. Unlike the viral genome at the pre-replication stage, most of the replicating HSV-1 genome is in a nucleosome-free state [24], and likely assumes a less condensed structure. The nucleosome-like HSV-1 genome is unstable and the accessibility to micrococcal nuclease (MNase) changes throughout the replication process. HSV-1 DNA is quantitatively recovered in complexes fractionating as mono- to polynucleosomes from nuclei harvested at 2, 5, 7, or 9 h post-infection. At 1 h post-infection, the whole HSV-1 genome is in nucleosomal stage and, at 2, 5, 7, or 9 h post-infection the viral genome lose nucleosome in different levels, suggested the stability of HSV-1 DNA nucleosomal complexes changes throughout the lytic infection cycle [5, 18, 24, 25]. To directly observe the dynamic structural changes in the HSV-1 replication process, probes were designed to recognize the termini of the viral genome (Fig. 2a). The two probes were labeled with either DIG or Biotin to give them two different colors.

Fig. 2 Replication renders compact HSV-1 genomes into relaxed structures. All cells were infected with HSV-1 17+ strain for 6 h, then prepared for FISH. In first line, signals are captured from red channel, which were hybridized with Biotin labeled probe; Second line, signals are captured from green channel, which were hybridized with DIG labeled probe; Third line, images are merged to examine the colocalization situation of two color signals; Fourth line, partial enlarged detail of figures in the third line are shown; Fifth line, images from the third line were analyzed, which were done with Image-Pro Plus 6.0 sofrware (USA). a: A brief description of HSV-1 genome structure. Relative to HSV-1 genome, red probe labeled with Biotin locates at the right terminal, which contains IR S , TR S and U S region (according to NC_001806.2 127235–131131, 132647–133909, 134056–134931, 135225–136670, 136747–137463, 138423–139607, 139789–140961, 141247–142899 and 147066–150962). Green probe labeled with DIG locates at the right terminal, which contains TR L , IR L and partial U L region (according to NC_001806.2, 513–1259, 2262–2318, 3084–3750, 3887–5490, 9338–10012, 10991–11665, 12484–15132, 151131–17161, 18225–20477, 20705–23260, 120884–122487, 122624–123290, 124056–124112 and 125115–125861). b-g: Cells were infected at a MOI of 0.1 PFU/cell. At early stage of HSV-1 replication, images are captured with STED microscopy and then analyzed. h-m: Cells were infected at a MOI of 5 PFU/cell. At late stage of HSV-1 replication, images are captured with STED microscopy and then analyzed. Host cell nucleus are indicated with white dotted lines. e, f, k, l: Higher zooms of regions inside the white rectangles are shown. Scale bars, 2.5 μm. Rr: correlation coefficient; R: overlapping coefficient; k: antigen contribution Full size image

BJ cells were infected at a low MOI of 0.1 PFU/cell and were processed for STED microscopy at the early stage of viral replication. Figure 2b (red) and 2C (green) are merged in Fig. 2d to show how the two colored signals relate. Pearson analysis of Fig. 2g shows that most of signals overlapped under STED microscopy (Fig. 2d), The Rr and R are 0.622 and 62.7 % (Fig. 2g), respectively. Parts of Fig. 2d (white rectangles) are enlarged to reveal two typical examples (Fig. 2e, f), where the red and green signals are directly connected or overlap. As Fig. 2e shows, the green signal is connected with the red oblong signal, but in Fig. 2f, the two colors sit right on top of each other. This is likely a result of differences in viral genome orientation. Compared with the correlation between two colored probes directed to the same region of the viral genome, the two probes directed toward different regions of the viral genome shows significantly lower correlation than the probes from the same region (compare Fig. 1h, k and 2d, g). The average distance between the two color signals from the same probe is 41.9 nm, but that of different probes is 111.9 nm, 2.7-fold higher (Fig. 3). These results suggest that STED microscopy is able to distinguish different regions of the viral genome at early stage of replication.

Fig. 3 Average distances of the same probe and different probes. Distances of the same probe and different probes were calculated under STED microscopy. The average distance of the same probe is 41.9 nm and that of different probes is 111.9 nm, which is 2.7-fold higher than the same probe, p value < 0.001 (***). The data were evaluated with the Students’ t-test Full size image

We next measured the distance between the different regions of HSV-1 genome in fully developed replication compartments. Signals in Fig. 2h (red) and Fig. 2i (green) are merged in Fig. 2j, and Pearson analysis is shown in Fig. 2m. Unlike the early stage of replication, viral genomes in advanced replication compartments do not show overlap and display very low correlation between the red and green signals (Fig. 2j). The Rr and R of Fig. 2j are 0.121 and 21.6 % (Fig. 2m), respectively, indicating very low correlations. Parts of Fig. 2j, which are indicated by white rectangles, are enlarged to reveal two typical examples (Fig. 2k, l), where we could see that the red and green probes detected elongated, fiber like structures.

In Fig. 3, the average distance between the two color signals from the same probe is 41.9 nm with a range from 22.6 nm to 70.8 nm, where as that of different probes is 111.9 nm with a range from 81.4 nm to 167.6 nm. At the pre-replication stage or early stage of replication, both the distances between the two color probes directed towards the same region, and the two probes, directed to different regions are relatively small. But, as viral replication progresses, these distances become greater. These results (Figs. 1, 2 and 3) suggest that pre-replication and early replication HSV-1 genomes exist as compact structures, while viral genomes in later replication compartments assume relaxed structures occupying significantly large space.

The ICP8 signals is highly related to the replicating HSV-1 genome

ICP8 interacts with the replicating parts of the viral genome and is used as a marker of HSV-1 replication. It also possesses multiple functions to facilitate viral replication and regulate viral genes expression [20, 22, 38, 39]. We therefore examined the distribution of ICP8 during replication to reveal the dynamic changes in the HSV-1 genomes.

Again, BJ cells were infected at a high MOI of 5 PFU/cell for 6 h and HSV-1 genomes were detected by FISH using labeled BAC clone probe covering the entire HSV-1 genome. As shown in the analysis in Fig. 4, ICP8 IF signals are tightly colocalized or associated with HSV-1 genome at both early (Fig. 4c) and late stages of replication (Fig. 4i). Colocalization coefficient (m2) describes contribution of positive staining pixels from each selected channels [36]. The value of m2 in Fig. 4c and Fig. 4i are 0.999 for both (Fig. 4f, l), indicating that 99.9 % green (ICP8) colocalize with red pixels (HSV-1 genome) in these figures. Figure 4d and e show local enlargements of the two white squares (Fig. 4c) to reveal visually the red and green signals are closely associated. As viral replication compartments became larger, ICP8 positive areas also grew with the compartments to eventually occupy the whole host nucleus (Fig. 4h). While the Rr and R of early stage of replication are 0.273 and 59.1 %, respectively, those of late stage of replication are 0.339 and 51.5 %, respectively. From a comparison between Fig. 4d and j, we could note an increase of viral genome signals and a reduction of ICP8 signals. This is because, at the early stage of replication, the infected nucleus has a large reserve of ICP8 proteins to prepare for replication, and viral genomes are in a smaller number. While, at the late stage of replication, the situation is reversed, with a huge number of viral genomes and a relative smaller amount of ICP8 proteins in the host cell nucleus. Consequently, at the early stage, the Rr value is lower than that at late stage of replication. With the development of replication compartments, the structure of the viral genome becomes more and more relaxed, and the average distance between ICP8 protein and the HSV-1 genome changes from 132.4 nm to 183.6 nm, p value < 0.001 (Fig. 7). Thus, R decreases with the replication progress from early to late stage.

Fig. 4 ICP8 signals is highly related to the replicating HSV-1 genome. All cells were infected with HSV-1 17+ strain and at a MOI of 5 PFU/cell for 6 h, then prepared for IF-FISH. a-c: At early stage of HSV-1 replication, images are captured with STED microscopy. d, e: Higher zooms of regions inside C are shown, which are indicated by white squares. f: Analysis results of C is shown. g-i: At late stage of HSV-1 replication, images are captured with STED microscopy. j, k: Higher zooms of regions inside I are shown, which are indicated by white squares. l: Analysis results of I is shown. Host cell nucleus are indicated with white dotted lines. Scale bars, 2.5 μm. Rr: correlation coefficient; R: overlapping coefficient; m2: colocalization coefficient Full size image

ICP8 occupies sub-structures within the viral replication compartments distinct from host RNA Pol II

Molecular and immunofluorescent studies suggest that HSV-1 replication and viral gene transcription are both occurring within the viral replication compartments [40]. However, transcription and DNA replication are two incompatible processes, i.e. the same region of the genome is difficult to replicate and transcribe at the same time [41]. Viral proteins for HSV-1 replication and viral genes are all transcribed by host RNA Pol II [42, 43]. RNA Pol II is regulated by phosphorylation of its carboxyl-terminal domain (CTD), with modification occurring primarily on serine 2 and 5 of the CTD. The serine 2 phosphorylated form of RNA Pol II (RNA Pol II Ser2P) is mostly associated with elongating form and active transcription, while the serine 5 phosphorylated form (RNA Pol II Ser5P) is more related to paused polymerase [44].

To determine how the ICP8 staining signals is related to RNA Pol II, we firstly performed double immunostaining using anti-ICP8 monoclonal antibody (Fig. 5a, d, i) and anti-RNA Pol II Ser2P polyclonal antibody (Fig. 5b, e, j). The images are merged to examine the colocalization of two color signals. As shown in Fig. 5f, there is a slight but visible increase of the RNA Pol II Ser2P colocalized with ICP8 marked early replication compartments. Local enlargement (Fig. 5g) shows that these two signals are related but do not overlap. The Rr and R of Fig. 5f are 0.404 and 66.9 % (Fig. 5h), respectively.

Fig. 5 Double immunostaining of ICP8 and RNA Pol II Ser2P. Experimental group cells were infected with HSV-1 17+ strain for 6 h, then fixed for IF. In first line, signals are captured from red channel, which were stained with anti-ICP8 monoclonal antibody; Second line, signals are captured from green channel, which were stained with anti-RNA Pol II Ser2P polyclonal antibody; Third line, images are merged to examine colocalization situation of two color signals; Fourth line, partial enlarged detail of figures in the third line are shown; Fifth line, images from the third line were analyzed, which were done with Image-Pro Plus 6.0 software (USA). a-c: Cells were not infected, images are captured with STED microscopy. d-h: Cells were infected at a MOI of 0.1 PFU/cell, images are captured with STED microscopy and then analyzed. i-m: Cells were infected at a MOI of 5 PFU/cell, images are captured with STED microscopy and then analyzed. Host cell nucleus are indicated with white dotted lines. g, l: Higher zooms of regions inside the white squares are shown. Scale bars, 2.5 μm. Rr: correlation coefficient; R: overlapping coefficient Full size image

To observe well developed replication compartments, cells were infected at a high MOI of 5 PFU/cell for 6 h prior to fixing for IF analysis. In these cells (Fig. 5i), RNA Pol II Ser2P evenly distributed, with a slight enrichment in areas overlapping with the ICP8 labeled replication compartments (Fig. 5j). Again ICP8 and RNA Pol II Ser2P do not show obvious overlap (Fig. 5k). The Rr value of Fig. 5k is 0.268, and the R value is 60.1 % (Fig. 5m). The average distances between ICP8 and RNA Pol II Ser2P at early and late stages of replication are 262.2 nm and 283.0 nm, respectively, and the difference between these two is not significant, p value > 0.05 (Fig. 7). These results suggest that ICP8 and RNA Pol II Ser2P do not show significant association.

ICP8 and RNA Pol II Ser5P double staining were conducted, but unlike RNA Pol II Ser2P, RNA Pol II Ser5P showed stronger colocalization in the viral replication compartments at 6 h post-infection at a low MOI of 0.1 PFU/cell and at the early stage of replication (Fig. 6f). The Rr and R of Fig. 6f are 0.464 and 56.2 % (Fig. 6h), respectively. When cells were infected at a high MOI of 5 PFU/cell and at the late stage of replication, RNA Pol II Ser5P still colocalizes with ICP8 (Fig. 6k). The Rr and R of Fig. 6k are 0.333 and 56.2 % (Fig. 6m), respectively.

Fig. 6 Double immunostaining of ICP8 and RNA Pol II Ser5P. Experimental group cells were infected with HSV-1 17+ strain for 6 h, then fixed for IF. In first line, signals are captured from red channel, which were stained with anti-ICP8 monoclonal antibody; Second line, signals are captured from green channel, which were stained with anti-RNA Pol II Ser5P polyclonal antibody; Third line, images are merged to examine colocalization situation of two color signals; Fourth line, partial enlarged detail of figures in the third line are shown; Fifth line, images from the third line were analyzed, which were done with Image-Pro Plus 6.0 software (USA). a-c: Cells were not infected, images are captured with STED microscopy. d-h: Cells were infected at a MOI of 0.1 PFU/cell, images are captured with STED microscopy and then analyzed. i-m: Cells were infected at a MOI of 5 PFU/cell, images are captured with STED microscopy and then analyzed. Host cell nucleus are indicated with white dotted lines. g, l: Higher zooms of regions inside the white squares are shown. Scale bars, 2.5 μm. Rr: correlation coefficient; R: overlapping coefficient Full size image

When viral replication switches from early to late stage, the average distances between ICP8 and RNA Pol II Ser5P change from 195.7 nm to 247.0 nm, with a p value < 0.001 (Fig. 7). This distance is smaller than the distance between ICP8 and RNA Pol II Ser2P (Fig. 7, p value < 0.05), suggesting ICP8 is positioned closer to RNA Pol II Ser5P than Ser2P. When comparing these values with average distance between ICP8 and viral genome, we found that the distance between ICP8 and HSV-1 genome is always closer than that of ICP8 and RNA Pol II. These differences suggest that viral replication and transcription are partitioned into distinct sub-structures within the replication compartments.