Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis

Reviewer #1 (Public review):

Summary:

Nahas et al. investigated the roles of herpes simplex virus 1 (HSV-1) structural proteins using correlative cryo-light microscopy and soft X-ray tomography. The authors generated nine viral variants with deletions or mutations in genes encoding structural proteins. They employed a chemical fixation-free approach to study native-like events during viral assembly, enabling observation of a wider field of view compared to cryo-ET. The study effectively combined virology, cell biology, and structural biology to investigate the roles of viral proteins in virus assembly and budding.

Strengths:

(1) The study presented a novel approach to studying viral assembly in cellulo.

(2) The authors generated nine mutant viruses to investigate the roles of essential proteins in nuclear egress and cytoplasmic envelopment.

(3) The use of correlative imaging with cryoSIM and cryoSXT allowed for the study of viral assembly in a near-native state and in 3D.

(4) The study identified the roles of VP16, pUL16, pUL21, pUL34, and pUS3 in nuclear egress.

(5) The authors demonstrated that deletion of VP16, pUL11, gE, pUL51, or gK inhibits cytoplasmic envelopment.

(6) The manuscript is well-written, clearly describing findings, methods, and experimental design.

(7) The figures and data presentation are of good quality.

(8) The study effectively correlated light microscopy and X-ray tomography to follow virus assembly, providing a valuable approach for studying other viruses and cellular events.

(9) The research is a valuable starting point for investigating viral assembly using more sophisticated methods like cryo-ET with FIB-milling.

(10) The study proposes a detailed assembly mechanism and tracks the contributions of studied proteins to the assembly process.

(11) The study includes all necessary controls and tests for the influence of fluorescent proteins.

Weaknesses:

Overall, the manuscript does not have any major weaknesses, just a few minor comments:

(1) The gel quality in Figure 1 is inconsistent for different samples, with some bands not well resolved (e.g., for pUL11, GAPDH, or pUL20).

(2) The manuscript would benefit from a summary figure or table to concisely present the findings for each protein. It is a large body of manuscript, and a summary figure showing the discovered function would be great.

(3) Figure 2 lacks clarity on the type of error bars used (range, standard error, or standard deviation). It says, however, range, and just checking if this is what the authors meant.

(4) The manuscript could be improved by including details on how the plasma membrane boundary was estimated from the saturated gM-mCherry signal. An additional supplementary figure with the data showing the saturation used for the boundary definition would be helpful.

(5) Additional information or supplementary figures on the mask used to filter the YFP signal for Figure 4 would be helpful.

(6) The figure legends could include information about which samples are used for comparison for significance calculations. As the color of the brackets is different from the compared values (dUL34), it would be great to have this information in the figure legend.

(7) In Figure 5B, the association between YFP and mCherry signals is difficult to assess due to the abundance of mCherry signal; single-channel and combined images might improve visualization.

(8) In Figure 6D, staining for tubulin could help identify the cytoskeleton structures involved in the observed virus arrays.

(9) It is unclear in Figure 6D if the microtubule-associated capsids are with the gM envelope or not, as the signal from mCherry is quite weak. It could be made clearer with the split signals to assess the presence of both viral components.

(10) The representation of voxel intensity in Figure 8 is somewhat confusing. Reversion of the voxel intensity representation to align brighter values with higher absorption, which would simplify interpretation.

(11) The visualization in panel I of Figure 8 might benefit from a more divergent colormap to better show the variation in X-ray absorbance.

(12) Figure 9 would be enhanced by images showing the different virus sizes measured for the comparative study, which would help assess the size differences between different assembly stages.

Overall, this is an excellent manuscript and an enjoyable read. It would be interesting to see this approach applied to the study of other viruses, providing valuable insights before progressing to high-resolution methods.

Reviewer #2 (Public review):

Summary:

For centuries, humans have been developing methods to see ever smaller objects, such as cells and their contents. This has included studies of viruses and their interactions with host cells during processes extending from virion structure to the complex interactions between viruses and their host cells: virion entry, virus replication and virion assembly, and release of newly constructed virions. Recent developments have enabled simultaneous application of fluorescence-based detection and intracellular localization of molecules of interest in the context of sub-micron resolution imaging of cellular structures by electron microscopy.

The submission by Nahas et al., extends the state-of-the-art for visualization of important aspects of herpesvirus (HSV-1 in this instance) virion morphogenesis, a complex process that involves virus genome replication, and capsid assembly and filling in the nucleus, transport of the nascent nucleocapsid and some associated tegument proteins through the inner and outer nuclear membranes to the cytoplasm, orderly association of several thousand mostly viral proteins with the capsid to form the virion's tegument, envelopment of the tegumented capsid at a virus-tweaked secretory vesicle or at the plasma membrane, and release of mature virions at the plasma membrane.

In this groundbreaking study, cells infected with HSV-1 mutants that express fluorescently tagged versions of capsid (eYFP-VP26) and tegument (gM-mCherry) proteins were visualized with 3D correlative structured illumination microscopy and X-ray tomography. The maturation and egress pathways thus illuminated were studied further in infections with fluorescently tagged viruses lacking one of nine viral proteins.

Strengths:

This outstanding paper meets the journal's definitions of Landmark, Fundamental, Important, Valuable, and Useful. The work is also Exceptional, Compelling, Convincing, and Solid. The work is a tour de force of classical and state-of-the-art molecular and cellular virology. Beautiful images accompanied by appropriate statistical analyses and excellent figures. The numerous complex issues addressed are explained in a clear and coordinated manner; the sum of what was learned is greater than the sum of the parts. Impacts go well beyond cytomegalovirus and the rest of the herpesviruses, to other viruses and cell biology in general.

Weaknesses:

I have a few suggestions for minor adjustments in the text.

Reviewer #3 (Public review):

Summary:

Kamal L. Nahas et al. demonstrated that pUL16, pUL21, pUL34, VP16, and pUS3 are involved in the egress of the capsids from the nucleous, since mutant viruses ΔpUL16, ΔpUL21, ΔUL34, ΔVP16, and ΔUS3 HSV-1 show nuclear egress attenuation determined by measuring the nuclear:cytoplasmic ratio of the capsids, the dfParental, or the mutants. Then, they showed that gM-mCherry+ endomembrane association and capsid clustering were different in pUL11, pUL51, gE, gK, and VP16 mutants. Furthermore, the 3D view of cytoplasmic budding events suggests an envelopment mechanism where capsid budding into spherical/ellipsoidal vesicles drives the envelopment.

Strengths:

The authors employed both structured illumination microscopy and cellular ultrastructure analysis to examine the same infected cells, using cryo-soft-X-ray tomography to capture images. This combination, set here for the first time, enabled the authors to obtain holistic data regarding a biological process, as a viral assembly. Using this approach, the researchers studied various stages of HSV-1 assembly. For this, they constructed a dual-fluorescently labelled recombinant virus, consisting of eYFP-tagged capsids and mCherry-tagged envelopes, allowing for the independent identification of both unenveloped and enveloped particles. They then constructed nine mutants, each targeting a single viral protein known to be involved in nuclear egress and envelopment in the cytoplasm, using this dual-fluorescent as the parental one. The experimental setting, both the microscopic and the virological, is robust and well-controlled. The manuscript is well-written, and the data generated is robust and consistent with previous observations made in the field.

Weaknesses:

It would be helpful to find out what role the targeted proteins play in nuclear egress or envelopment acquisition in a different orthoherpesvirus, like HSV-2. This would confirm the suitability of the technical approach set and would also act as a way to validate their mechanism at least in one additional herpesvirus beyond HSV-1. So, using the current manuscript as a starting point and for future studies, it would be advisable to focus on the protein functions of other viruses and compare them.