(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



Infection-induced chromatin modifications facilitate translocation of herpes simplex virus capsids to the inner nuclear membrane

['Vesa Aho', 'Department Of Biological', 'Environmental Science', 'Nanoscience Center', 'University Of Jyväskylä', 'Jyväskylä', 'Sami Salminen', 'Salla Mattola', 'Alka Gupta', 'Felix Flomm']

Date: 2022-02 

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.

Herpes simplex virus type 1 is a DNA virus that is studied intensively due to its high prevalence in humans, its ability to cause many diseases upon lytic replication or to evade the immune system in a latent state, and its potential use in oncolytic and immunotherapeutic applications. The nuclear replication of herpes simplex virus type 1 leads to the emergence of viral replication compartments together with relocalization and condensation of the host cell chromatin to the nuclear periphery. The reorganization of the host chromatin potentially hampers the intranuclear transport and nuclear egress of newly assembled progeny capsids. Nuclear capsids move by diffusion prior to their viral nuclear egress complex-mediated exit through the nuclear envelope. However, it is not understood how the capsids travel through the chromatin network. Our results show that infection-induced changes in chromatin architecture enhance nuclear diffusion of capsids at late infection, and that this transport is the rate-limiting step in the nuclear egress of the virus. Understanding the kinetics of capsid translocation in the nucleus is of fundamental interest in the field of DNA virus research because it explains how viruses are able to move in the chromatin before their nuclear egress.

Although the virus-induced modulation of the chromatin architecture and the capsid kinetics are increasingly understood, the mechanisms of capsids motion inside the infection-modified chromatin network have not been characterized. Herein, we have tracked fluorescently tagged HSV-1 capsids in living cells and correlated the intranuclear capsid localization and motion with the local chromatin environment. The progeny capsids were concentrated in the VRC-incorporating chromatin-empty regions, and the size of these regions increased as the infection progressed. The capsid density in the chromatin-empty regions was always higher than in the chromatin regions. Even though there was almost twofold increase in the number of detected capsids from 4 to 8 hpi, the capsid density in the chromatin increased only later at 12 hpi. Moreover, our analyses of the trajectories of individual capsids indicated that they were more likely to enter chromatin regions at 12 hpi than at 8 hpi. These data suggest that the chromatin restricted capsid motion as it was marginalized at 8 hpi. Later the permeability of chromatin was increased, which would facilitate the capsids to access the nuclear envelope. We further observed that capsids diffused faster in the chromatin-empty than in the chromatin regions, and that in both the diffusion rate increased as the infection progressed. At no time point was there accumulation of capsids at the nuclear envelope, which indicates that capsid translocation through the chromatin is a slower process than the transport through the nuclear envelope. Visualization of infected cells with electron microscopy confirmed the low density of capsids at the nuclear envelope and further showed that the number of DNA-containing capsids in the nucleus was relatively low, which may be an additional reason for the low density of capsids at the nuclear envelope since the DNA-containing capsids are preferentially retained there for budding. Altogether, these results imply that the nuclear environment undergoes changes during HSV-1 infection that modulate intranuclear motion and localization of capsids.

While the dynamics of cytoplasmic HSV-1 capsids have been investigated in detail [ 28 ], nuclear diffusion and its restriction by the chromatin network has been considered only recently [ 19 , 20 , 29 , 30 ]. Herpesvirus capsids are relatively large particles for intracellular transport; microinjected dextran beads of 110 nm, similar in size to HSV-1 capsids, are immobile in the nuclei of non-infected cells [ 31 ]. However, recent studies have shown that HSV-1 capsids diffuse inside the interchromatin domains that enlarge during the infection [ 20 ]. As the infection progresses further, the growing VRCs containing most of the progeny capsids displace the host chromatin toward the nuclear envelope and the center of the nucleus becomes mostly devoid of chromatin [ 29 , 30 , 32 ]. Previous studies have implied that VRCs are molecularly crowded and dynamic structures with temporal changes in their biomolecular composition [ 10 , 33 – 35 ]. As the chromatin is marginalized to the nuclear periphery, the chromatin-empty regions located in the central parts of the nucleus remain enriched with virus capsids. The displaced chromatin accumulates underneath the nuclear envelope as a layer that is most concentrated around 8 hours post infection (hpi), but subsequently low-density regions and channels that traverse the marginalized chromatin appear, which likely guide the capsids to the nuclear envelope [ 30 , 36 ].

Enveloped herpesviruses are widespread and important human pathogens. Herpes simplex virus 1 (HSV-1), a causative agent of the common cold sores, is a virus with a DNA-containing icosahedral capsid of ~125 nm in diameter [ 1 ]. Upon entering host cells, the viral capsids must travel to the nuclear envelope for docking to the nuclear pores where they expel their genomes into the nucleus [ 2 – 4 ]. The early studies of HSV-1 capsid motion in the cytoplasm elucidated that the motion to the nuclear envelope occurs via microtubules [ 5 – 7 ]. After capsid association with the nuclear pore complex, the viral genomes are injected into the nucleus and the formation of viral replication compartments (VRCs) is initiated. VRCs are dedicated sites for virus genome replication, and recent studies have revealed that each VRC originates from one individual viral genome [ 8 – 12 ]. As the infection progresses, the VRCs expand and the likelihood of their fusion and formation of enlarged compartments increases [ 4 , 13 ]. VRCs become enriched with capsid proteins and capsids as the infection progresses [ 14 , 15 ], but whether capsid assembly and packaging occur within the VRCs or whether the capsids accumulate at them later is not clear. However, capsids need to travel from the VRCs to the nuclear border for egress through the nuclear envelope into the cytosol [ 16 – 18 ]. The relatively large capsids with a diameter of 125 nm move through the nucleoplasm by diffusion [ 19 , 20 ]. Due to their large size, the capsids cannot pass through the nuclear pores, but instead bud through the nuclear envelope utilizing the nuclear egress complex formed by viral proteins UL31 and UL34 [ 21 – 23 ]. This process favors nucleocapsids, and most of the cytoplasmic capsids are DNA containing even though in the nucleus they are the in the minority [ 24 , 25 ]. After envelopment of the capsid at the inner nuclear membrane and subsequent envelope fusion at the outer membrane, capsids are released into the cytosol and transported to the trans-Golgi network or an endosomal compartment for final envelopment before they are released from the cell by exocytosis [ 18 , 26 , 27 ].

(A) A transmission electron microscope image of a cell infected 12 h prior to sample fixation. The capsids have been labeled as type A (empty capsids, marked with white circles), type B (capsids containing the protein scaffold, black circles) and type C (DNA-containing nucleocapsids, blue circles). The area marked with a white square is magnified at the lower right corner of the image, showing each of the capsid types. The scale bar represents 2 μm (200 nm in the magnified region). (B) The combined density of every capsid type as a function of the distance from the outer nuclear membrane. (C) The number of each capsid type divided by the number of all capsids (blue), and the fraction of each capsid type that is located within 1 μm from the nuclear envelope (gray). The shaded regions and error bars show the standard error of the mean. The significance values shown inside the bars are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). The letter after the significance symbol indicates the capsid type that the value was compared with. The sample size was N = 11 cells.

To verify the low density of capsids at the nuclear envelope and to see if the localization of capsids in the nucleus depends on their stage of DNA packaging, we used electron microscopy to image cells infected 12 h before sample fixation. Visual observation revealed that relatively few capsids were located close to the nuclear envelope or were in the process of budding through it. In electron microscopy micrographs three types of capsids can be distinguished: empty capsids (type A), capsids that contain the inner protein scaffold (type B) and capsids that contain the viral genome (type C). We manually annotated capsids by their type based on their appearance ( Fig 4A ). Analysis of capsid density as a function of distance from the outer nuclear membrane verified that the number of capsids was lowest near the nuclear membrane ( Fig 4B ). We calculated the prevalence of each capsid type in the nucleus ( Fig 4C ), and the B type was the most common with the value of 72 ± 4%. The portion of A capsids was 9.3 ± 1.2% and C capsids 19 ± 4%. Between 14% and 17% of each capsid type was located within 1 μm from the outer nuclear membrane, and there was no statistically significant difference between the capsid types ( Fig 4C ).

(A) The mean density of capsids as a function of distance from the nuclear envelope summed over the time series. The negative x-axis values show the distance to the cytoplasmic side and positive x-axis values the distance to the nucleoplasmic side of the nuclear border. (B) The change in the shortest distance to the nuclear border during a capsid track. On the positive x-axis the capsid moves away from the nuclear border during a track and on the negative x-axis toward it. (C) The fraction of the area within 1 μm from the outer edge of the chromatin visited by capsids during 40 s. (D) The mean number of capsids that translocated from within the chromatin to outside of it at the edge of the nucleus. (E) An image series showing a capsid approaching the edge of the chromatin at the border of the nucleus and passing through it. The error bars show the SEM. The significance values shown inside the bars are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). The number after the significance symbol indicates the infection time point that the value was compared with. For every time point the sample size was 28 cells. The scale bar represents 5 μm.

In searching for factors that might modulate capsid egress, we examined capsid localization and motion relative to the nuclear border. To determine whether egressing capsids were retained at the nuclear envelope, we determined the density of capsids as a function of distance from the nuclear border on both the nuclear and cytoplasmic sides of it ( Fig 3A ). Interestingly, we did not detect any capsid accumulation near the nucleus-cytoplasm interface. Next, we tested whether capsid motion was directed toward the nuclear envelope by comparing the distance to the nuclear border at the beginning and the end of each capsid track. At each time point, the histograms were quite symmetrical with respect to whether the capsids were moving toward or away from the nuclear border, indicating that there was no preferred direction with respect to the nuclear envelope ( Fig 3B ). To characterize the capsid movements toward the nuclear envelope, we calculated the proportion of the region within 1 μm from the outer edge of the chromatin that had a capsid visiting them during the imaged time course. At 12 hpi, the capsids were able to access a proportionally larger region than at 4 or 8 hpi ( Fig 3C ). Finally, we determined the number of capsids that had started their tracks within a chromatin region and were able to leave it at the border of the nucleus ( Fig 3D and 3E ). Our data show that the number of capsids traveling through the chromatin regions increased steadily. At 12 hpi, the number was highest, but there was also a large variation between cells.

Taken together, the difference in the crossing probability between chromatin and chromatin-empty regions at 8 and 12 hpi is consistent with the notion that capsid motion is restricted by chromatin. However, the increase in the capsid diffusion coefficient in chromatin during the infection suggests that density of chromatin decreases as the infection progresses. The same appears to be true for the chromatin-empty regions. It should be also noted that the transport probability between chromatin and chromatin-empty regions was affected by the infection-induced changes.

As there was a clear difference in the capsid density between chromatin and the chromatin-empty regions, we analyzed if the chromatin density within the segmented chromatin affected the occurrence of capsids at those locations. The low-density pixels had a high probability of capsid detection, but surprisingly the effect was not very strong ( Fig 2E ). Even at high chromatin densities there was a relatively high probability to find capsids in those regions. The total size of the regions with such high chromatin fluorescence intensity values was, however, very small. Finally, to gain deeper understanding on restrictions to capsid movement, the number and locations of immobile capsids were analyzed ( Fig 2F ). At every infection time point the fraction of immobile capsids in chromatin was higher than in chromatin-empty regions. However, the variations were so large that no statistically significant differences could be established.

To examine the motion of capsids between the nuclear regions, we analyzed the probability of a capsid starting on either side near the border between chromatin and chromatin-empty regions and moving to the other side ( Fig 2D ). The probability to cross the interface from chromatin-empty regions to chromatin decreased from 4 to 8 hpi, but this decrease was not statistically significant (p>0.05). However, from 8 to 12 hpi, there was a significant increase in the crossing probability (p<0.05). This is in line with the chromatin accessibility data described above and with our previous study indicating that marginalized chromatin is denser at 8 hpi after which the density decreases at 12 hpi [ 30 ].

The MSD value where the curve saturates can give indication of the size of the compartments where the particles diffuse in. The actual saturation values were difficult to determine due to large uncertainties of the curves (which probably result from large variations in the sizes of confining regions and from the lower number of tracks extending to longer times). The size of the diffusion compartments seemed to be, however, from about 1.0 μm to 1.4 μm when analyzed using equation , where MSD sat is the saturation value of the MSD and d HSV is the capsid diameter of 125 nm [ 20 ].

The motion of capsids appeared to be subdiffusive (α < 1): the mean anomalous exponent was between 0.69 and 0.90 in both regions at every measured infection time point. At any given time point, the diffusion coefficient was higher in the chromatin-empty than in the chromatin regions, and the ratio of the diffusion coefficients between the regions was lowest at 4 hpi and highest at 8 hpi. Interestingly, both in the chromatin and chromatin-empty regions the diffusion coefficient increased as the infection progressed. In chromatin the difference between 4 and 8 hpi was not very large, but at 12 hpi a bigger change occurred. In the chromatin-empty regions there was a steadier increase from 4 to 12 hpi.

(A) Capsid tracks during 10 seconds in an HSV-1 infected cell. Only tracks that were at least 5 frames (0.5 s) long are shown. Cytoplasmic tracks are also shown, but they were excluded from the analyses. The stained chromatin is shown in gray. (B) The mean squared displacement (MSD) of nuclear capsids in chromatin and chromatin-empty regions as a function of time. (C) The mean fraction of chromatin and chromatin-empty regions visited by capsids during 40 s. (D) The mean probability of a capsid starting on one side of the border of chromatin regions to move to the other side of a border during a capsid track. (E) The mean number of capsids detected in chromatin during 40 s as a function of chromatin density. The number of detections was normalized by the size of the detection area. (F) The ratio of immobile particles to the number of all particles in chromatin and chromatin-empty regions. The shaded areas around the data points and the error bars show the SEM. Statistical significances were determined using Student’s t-test. The significance values shown inside the bars are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). The number after the significance symbol indicates the infection time point that the value was compared with. Values were compared for the same region at different time points (indicated by a different time code than the time point of the bar) and for the different regions only within each time point (indicated by the same time code as the time point of the bar). For every time point the sample size was 28 cells. The scale bar represents 5 μm.

The capsids were detected in each image, and their tracks were documented based on the individual capsid coordinates. The capsids seemed to move primarily in the low chromatin density regions, but a smaller fraction moved also within the denser chromatin regions at all three time points ( Fig 2A ). We examined capsid motion by correlating the extracted capsid coordinates with the segmented chromatin regions. We calculated the mean squared displacement (MSD) of capsids to determine how the chromatin affected the capsid mobilities during the course of the infection ( Fig 2B ). The curves had the characteristic shapes of confined diffusion, showing an initial rapid increase in the MSD and a final saturation due to particles being unable to penetrate boundaries of the confining regions. The saturation started to affect curves quite early, but at a short time scale the curves could be fitted with an equation for anomalous diffusion, D = 4Dt α , where D is the diffusion coefficient and α is the anomalous exponent ( S4 Fig ). The obtained diffusion coefficients and anomalous exponents at the time scale of 0–1.0 s are summarized in Table 1 .

(A) Spinning disk microscopy images of capsid distribution in Vero cells at 4, 8 and 12 hpi. Overlays of Hoechst-labeled chromatin (gray) and fluorescent capsid protein VP26-mCherry (green) are shown. (B) The mean relative intensity of Hoechst-stained nuclear chromatin as a function of the distance from the nuclear envelope at 4 (blue), 8 (orange) and 12 hpi (green). The shaded areas around the data points represent the SEM. (C) Hoechst-stained chromatin (gray) in an infected cell nucleus at 8 hpi and its automatic segmentation into chromatin (white) and chromatin-empty regions (black). (D) The mean area of segmented chromatin (dark gray) and chromatin-empty regions (light gray) and (E) the mean density of capsids in chromatin and chromatin-empty regions. The error bars show the SEM. Statistical significances were determined using Student’s t-test. The significance values shown inside the bars are denoted as ** (p<0.01), * (p<0.05) or ns (not significant). The number after the significance symbol indicates the infection time point that the value was compared with. Values were compared for the same region at different time points (indicated by a different time code than the time point of the bar) and for the different regions only within each time point (indicated by the same time code as the time point of the bar). For every time point the sample size was 28 cells. The scale bars represent 5 μm.

In summary, these results show that the chromatin-empty regions, which mostly correspond to the VRCs, grow as the chromatin is displaced toward the nuclear periphery. The capsid density was much higher in the chromatin-empty than in the chromatin regions from 4 to 12 hpi, which suggests that capsid motion was restricted by the chromatin regions. The capsid density ratio between chromatin and chromatin empty regions was highest at 8 hpi, indicating that chromatin was less accessible to capsids at this time point. At 12 hpi the chromatin accessibility was partially recovered.

To further assess the intranuclear localization of the HSV-1 capsids, the images were segmented and capsid coordinates recorded. The analysis revealed that the capsid density in the chromatin-empty regions increased from 4 to 12 hpi ( Fig 1E ). Even though the total number of capsids increased from 4 to 8 hpi, the density of capsids in the chromatin did not increase during this time. However, at 12 hpi an increase was detected. At every time point the capsid density was much higher in the chromatin-empty regions than in the chromatin regions. The capsid density ratio between the regions was 2.6 ± 0.4, 3.9 ± 0.5 and 2.8 ± 0.4 at 4, 8 and 12 hpi respectively.

Both the chromatin and the chromatin-empty regions are important regarding capsid transport. The chromatin-empty VRCs contain most of the newly assembled progeny capsids at any given time, and these capsids need to traverse the marginalized chromatin to get access to the nuclear envelope for egress into the cytosol. To gain more insight into the interplay of capsids and chromatin structure, we examined the capsid localization and later capsid motion in these regions by automatically segmenting the nucleus of each cell into chromatin and chromatin-empty regions (Figs 1C and S3 ). Throughout the infection, the chromatin cross-sectional area remained almost unaltered ( Fig 1D ). This also suggested that the segmentation of the chromatin regions was quite accurate. Importantly, the cross-sectional area of chromatin-empty regions increased more than twofold from 4 to 8 hpi, but then did not change significantly from 8 to 12 hpi.

The microscopy images indicated that the regions with low-intensity chromatin staining were located at the central parts of the nuclei and contained most of the viral capsids ( Fig 1A ). We also verified by a separate immunolabeling experiment that the low intensity region consisted mostly of VRC ( S1 Fig ). To quantify the infection-induced rearrangement of chromatin, we segmented the nuclei based on the DNA staining and examined the chromatin distribution with respect to the nuclear border ( Fig 1B ). The association of the chromatin border with the nuclear border was justified by the fact that chromatin is known to be separated from the nuclear envelope by the nuclear lamina, whose thickness is below the resolution of light microscopy ( S2 Fig ). The comparison of chromatin localization at various time points showed that the relative amount of DNA signal near the nuclear border increased from 4 to 8 hpi as the amount in the central parts decreased. From 8 to 12 hpi, the signal had shifted only slightly towards the nuclear border.

Discussion

The mechanisms that regulate the intranuclear transport of newly assembled, progeny HSV-1 capsids to the nuclear envelope are not well understood. It is known that capsids traverse the nucleoplasm by a diffusion-based mechanism, but it is not clear how the presence of intranuclear structures such as chromatin regions or VRCs affect this motion. Herein, single-particle tracking of capsids in the nuclei of living cells with labeled chromatin allowed us to detect capsid motion with respect to the chromatin environment.

The establishment of nuclear HSV-1 VRCs is accompanied by a marginalization of the host cell chromatin to the nuclear periphery [29,32]. Our analysis of the chromatin distribution as the HSV-1 infection progressed showed that this chromatin relocalization correlated with a concomitant increase in the sizes of chromatin-empty regions and the nucleus. As more capsids were formed from 4 to 8 hpi, the density of capsids increased in the chromatin-empty regions but not in the chromatin regions. However, at 12 hpi the capsid density increased in the chromatin regions. Moreover, the ratio of the capsid density in the chromatin-empty regions to the capsid density in the chromatin regions was highest at 8 hpi, which suggests that chromatin was less accessible to capsids at this intermediate time point. This temporal decrease and subsequent increase in chromatin accessibility is consistent with our previous expansion microscopy findings that the proportion of interchromatin spaces in the marginalized chromatin decreases from 4 to 8 hpi but increases again at 12 hpi [30]. Our results suggest that infection-induced changes in the chromatin accessibility, particularly the chromatin redistribution induced by the VRC expansion, and the increase of interchromatin spaces later in infection, may play a significant role in accessibility of the chromatin to viral capsids, and thereby influence the dynamics of the HSV-1 nuclear egress during the infection.

During interphase, the molecularly crowded nuclear environment with the chromatin network, subnuclear compartments and many proteins limits diffusion in the nucleoplasm [41,42]. Particularly, the three-dimensional organization of the chromatin modulates mobility and interactions of nuclear macromolecules [43–45]. The structural hindering of motion by the chromatin is dependent on size of the macromolecule [37,46–48]. For example, diffusion of larger structures such as mRNPs particles with a diameter of up to 25 nm is hindered by dense chromatin regions [37,49,50]. Moreover, the molecularly crowded cytoplasm or the compact chromatin act as diffusion barriers for viral capsids, e.g. HSV-1 capsids with a diameter of 125 nm [20,29,51]. In infected cells, the infection-induced compaction and marginalization of chromatin poses an additional challenge for the nuclear egress of HSV-1 capsids [30,36]. Here, we discovered that the capsids exhibited subdiffusional behavior, and that the slopes of the MSD(t)-curves decreased before they saturated in the time scale of seconds. The mean anomalous exponents were between 0.69 and 0.90. Subdiffusion is a sign of obstructed diffusion, and it has been observed for example for the intranuclear motion of adeno-associated viruses (α = 0.6–0.9, [52]) and telomeres (α = 0.32, [53]). Our analysis of the fitted diffusion coefficients demonstrated that diffusion was always more rapid in the chromatin-empty than in the chromatin regions. This is consistent with our soft x-ray microscopy studies showing that the density of biomolecules is significantly lower in the VRCs than in tightly packed chromatin [29]. The ratio of diffusion coefficient in the chromatin-empty regions to the diffusion coefficient in the chromatin regions was highest at 8 hpi, after which it decreased when the infection proceeded to 12 hpi. The strong increase in the diffusion rate from 8 to 12 hpi agrees with our previous study showing that the fraction of chromatin-free regions increases substantially in the marginalized chromatin when visualized with expansion microscopy [30]. As the infection progressed, the diffusion coefficients increased both in chromatin and in chromatin-empty regions. It is noteworthy that the chromatin-empty regions containing enlarged VRCs with viral genomes, viral proteins and capsids could be a crowded environment. Such a molecular crowding could hinder diffusion, however, our measurements demonstrate that there was no reduction in the diffusion coefficient or the anomalous exponent in the VRCs. This suggests that the VRCs do not become more crowded as the infection proceeds.

Our mean squared displacement analysis shows that diffusion in chromatin and chromatin-empty region was restricted to regions between 1.0 μm and 1.4 μm in size, which is in accordance with a previous study [20]. Surprisingly, the differences among the confinement regions of chromatin and chromatin-empty regions were quite small. Furthermore, as the confinement regions were smaller than the nuclei or the VRCs, the capsid motion was not, at least in our analysis time scale, mainly restricted by the nuclear envelope or the marginalized chromatin but by smaller subnuclear structures. Moreover, our analyses indicate that the probability of particles to translocate from chromatin-empty to chromatin regions increased from 8 to 12 hpi, which is consistent with our finding that capsid movement became faster in chromatin. It is possible that the crossing probability is not only affected by chromatin compaction, but by interface effects at the border of the chromatin. It has been shown even for much smaller particles than HSV-1 capsids that the particles may diffuse quite freely within low and high chromatin density areas but the motion between the two regions is more restricted [54]. There was no clear correlation between the chromatin density and the capsid localization. In general, the probability of capsid detection was high in the low chromatin density regions, but the capsids were found also in the higher density regions. Possibly we were unable to detect smaller channels of low chromatin density with the ~200 nm resolution in the lateral direction and below 500 nm in the axial direction. Notably, there were some high chromatin density pixels that had a high probability of capsid detection in them, which can possibly result from capsids being trapped in those regions. The presence of immobile capsids correlated with the progress of infection, and the highest fraction of immobile capsids were detected at early times post infection. Immobile capsids were located both in the chromatin-empty and chromatin regions; their static positions could be due to steric hindrance by a high chromatin density or by genome packaging into newly pre-assembled capsid shells [55–58]. Although we have here analyzed capsid motion only with respect to chromatin, the capsid motion can also be restricted by other nuclear bodies. However, these structures are typically small compared to the size of the chromatin regions, and they are also located at the central parts of the nucleus and therefore not blocking the capsid access to the nuclear envelope. For this reason, their effect on the capsid transport to the nuclear envelope is probably much smaller compared to that of the chromatin.

We did not observe any substantial accumulation of capsids beneath the nuclear envelope. This indicates that the time scale of capsid transport to the nuclear envelope is so slow compared to the nuclear egress that an accumulation does not happen. Therefore capsid transport through the marginalized chromatin seems to be a bottleneck of the nuclear egress of HSV-1 progeny capsids. Moreover, we observed that the number of DNA-containing capsids was quite low constituting approximately only one fifth of all the nuclear capsids. Since they are the favored capsid types in the nuclear egress [25], their low number, combined with the transport barrier caused by the marginalized chromatin, leads to their low density at the nuclear envelope. The tendency of capsids to diffuse similarly toward or away from the nuclear envelope was observed, and it suggests that the diffusion of capsids toward the nuclear envelope was not facilitated. The number of capsids that travelled from within chromatin to outside of it at the nuclear border increased as the infection progressed, but we did not observe those capsids moving a substantial distance away from the nucleus. The HSV-1 budding process at the inner nuclear membrane mediated by the nuclear egress complex takes, at least in vitro, more than five minutes [26], and is therefore a slower process than we could monitor in our experiments.

Advances in the camera technology allow imaging fluorescently-labelled capsids with high sensitivity and temporal resolution. The quantum efficiency of the latest CMOS cameras exceeds 90%, so there is not much room for improvement in that respect. With future advances in super-resolution microscopy, capsid-chromatin interaction can potentially be studied with even finer detail. When studying a population of infected cells, the variability in the state of infection among the individual cells can be a problem. Although the cells in our assays were isogenic, the heterogenous cellular microenvironment and the population context variability lead to asynchronicity in the viral entry, the initiation of viral transcription, and all subsequent steps of the HSV-1 life cycle despite an inoculation with a high MOI [59,60]. One of the challenges in the future studies will be to control this heterogenous progression of HSV-1 infection among individual cells, or more likely to take this into account. With automated imaging and analyses a higher number of cells can be analyzed, leading to more accurate measurements even in the case of large variations between cells. It should also be noted that the cell type or tissue specific chromatin environment of the host likely has an impact on capsid mobility and progression of infection, which needs to be addressed in the future studies.

By a thorough investigation of intranuclear capsid motions, we discovered the changing properties of the nuclear environment during HSV-1 infection. The Brownian motion of particles is determined by the temperature and viscosity of the medium, the size and shape of the particles, and the surrounding obstacles restricting the motion. Since the temperature in our experiments was kept constant and the tracked capsids are of constant size, the only variables were the viscosity of the nucleoplasm and the effects of obstacles. Therefore, the analysis of intranuclear viral capsid dynamics is a powerful tool to indirectly probe the viscous and obstructive properties of the nucleoplasm. Examining the capsid motion in living cells opens a window into understanding of the rules that drive the nuclear transport of viral progeny capsids throughout the nonhomogeneous nuclear environment during their egress. We believe that our study provides new insights into intranuclear motion of capsids and introduces novel ideas for studies of intranuclear transport.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010132

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/