Structural analysis of herpes simplex virus by optical super-resolution imaging

Structural analysis of herpes simplex virus by optical super-resolution imaging

The molecular structure of the genome of equine herpesvirus type 1 (EHV-1) was determined by restriction endonuclease mapping studies. There are eight known herpesviruses out of 100 known herpesviruses that infect humans. In HHV-6, DRL and DRR are identical and a sequences may therefore also occur at the U-DR junctions to give the arrangement aDRLa-U-aDRRa. Sequences within the viral cyclin N-terminus lock part of the cdk2 T-loop within the core of the complex. Nucleotide sequence analysis of this region revealed a single open reading frame of 1,515 base pairs. From this data, we propose a model of the protein organization inside the tegument. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn(2+) ions, two of which are located at the active site pocket.

One of the most widespread human members of this family, herpes simplex virus type-1 (HSV-1), is a common cause of cold sores but can also result in herpes keratitis1, a leading infectious cause of blindness, and herpes simplex encephalitis2, which has a high mortality even in patients treated with antiviral therapy. Corbin-Lickfett, K. The dsDNA endonuclease and packaging activities are performed by a protein complex, the terminase, composed by subunits UL56 and UL89 (4, 5). Electron microscopy (EM) and electron tomography (ET) studies of HSV-1 have revealed that virus particles have a spherical shape4, with diameter ranging from 155 to 240 nm, each containing an icosahedral capsid with a diameter of 125 nm5,6, which contains the viral DNA. The structure of the highly ordered capsid complex has been studied extensively and is now well defined. A partially ordered layer of tegument proteins surrounds the nucleocapsid and the viral envelope encloses the nucleocapsid/tegument core. The envelope carries a set of glycoproteins that are essential for virus entry and viral morphogenesis.

Cyclin homologues have been identified within the genomes of several γ-herpesviridae associated with neoplastic disorders, these include herpesvirus saimiri (HSV) (Albrecht et al., 1992; Jung et al., 1994), Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) (Chang et al., 1996; Russo et al., 1996a) and murine γ-herpesvirus 68 (Virgin et al., 1997). F. 8). In particular, VP1/2 (also known as pUL36) is the largest tegument protein (>330 kDa) and is essential for HSV-1 entry and assembly9,10. Owing to the interaction of its carboxy terminus with the minor capsid protein pUL25, VP1/2 is often referred to as being a part of the inner tegument. J. It encodes a two-domain 674 amino acid protein with predicted N-terminal ATPase and C-terminal nuclease activity (Fig.

VP16 also interacts with VP1/2 (ref. 14) and induces transcription of immediate-early viral genes, thus playing an important role in the viral life cycle15. Owing to its multiple interactions with other viral proteins, VP16 has been proposed to serve as a central organizer for the tegument12. Although it is now clear that the tegument is not as disordered as previously thought, its structure and organization are still poorly understood. The structures of two further cyclins, cyclin H and V-cyclin, have been determined in their unbound form, demonstrating that all three cyclins have similar three-dimensional folds despite their limited sequence similarity (Brown et al., 1995; Kim et al., 1996; Andersen et al., 1997; Schulze-Gahmen et al., 1999). EM studies of biological materials are often restricted to highly ordered structures and, therefore, cannot be used to resolve viral tegument and envelope organization. Moreover, although EM and ET are powerful tools to visualize overall physical structure, their ability to localize specific proteins is limited.

Optical techniques do not suffer this latter disadvantage; however, they are limited by optical diffraction, with a typical lateral resolution of ~200 nm. This resolution is comparable to the entire virus diameter and thus too coarse to reveal structural information. B. Similar results were obtained when the full-length gene was expressed in insect or mammalian cells. Although PALM20,23, stimulated-emission depletion24 or dSTORM25,26,27 have begun to be used in the investigation of viral replication and structure, their application in the context of virology research is still in its infancy. In particular, the lateral resolution achievable with SMLM (typically 20–30 nm, full width at half maximum) is well suited for the study of HSV-1 particles. The increased resolution in SMLM is based on the temporal separation and the subsequent precise localization of individual fluorescent emitters.

Structural analysis of herpes simplex virus by optical super-resolution imaging
Compared with other super-resolution methods, not only can the fluorophore localizations be used for the reconstruction of a high-resolution image, but they also allow for the counting of molecule numbers28,29,30, cluster analysis31,32 or geometric modelling33. One of the striking features of V-cyclin–cdk6 and K-cyclin–cdk6 complexes is their ability to evade inhibition by p21Cip/WAF and p27Kip. First, multi-colour localization microscopy was used to visually distinguish virus proteins in individual virus particles. We then designed a model-based analysis of SMLM data and used particle averaging (a method routinely used in EM34) to reconstruct a high-resolution image and to determine the position of individual protein layers within the virus particle with nanometre precision. We additionally investigated the effect of the fluorophore linker size and compared immuno-labelling strategies in the context of model-based analysis. Finally, our data set confirmed and quantified the spatial offset of the capsid with respect to the centre of the virion. A., Chen, I.

Other similar-sized constructs identified as partially soluble in small-scale testing did not maintain solubility during subsequent scale-up steps. We thus searched for a second fluorophore that could be reliably used in combination with AF647 for imaging virus particles and tested AF647/ATTO532, AF647/AF546 and AF647/AF568 in a range of photoswitching environments. All fluorophores were linked to secondary antibodies and imaging protocols were optimized for each fluorophore combination (). As shown in the top row in , for every fluorophore combination tested, two-colour dSTORM consistently demonstrated the envelope protein gD to be located at larger radial distance than the tegument protein VP16. The two tegument proteins VP16 and pUL37 showed a large overlap and similar radial distributions, suggesting that VP16 and pUL37 reside in the same region of the tegument layer (, bottom row). We observed an ∼50-fold increase in kinase activity using T160k2-phosphorylated cdk2 and M-cyclin (data not shown). However, by resolving and analysing individual virus particles, we could not consistently differentiate between outer and inner tegument, irrespective of dye combination used (, bottom row, second and third panel).

Next, we imaged virus particles within infected cells by using two-colour dSTORM. Here, to aid identification of the virus assembly state, we produced a fluorescently tagged recombinant HSV-1 that genetically encoded the small capsid protein VP26 fused to mTurquoise and the envelope protein gM fused to enhanced yellow fluorescent protein (EYFP). We were therefore able to locate individual capsids and discriminate between enveloped and non-enveloped particles within the infected cells. M. At both lateral edges of the β-sheet, β1 and β10 form short strands of only three amino acids each. The envelope protein gM (shown in yellow) was mostly present in the perinuclear region of the cytoplasm and near the plasma membrane, as expected for a viral envelope glycoprotein that undergoes vesicle-based transport through the secretory and endocytic compartments of the cell35. dSTORM imaging of both AF647 and AF568 allowed the identification and characterization of structural elements of individual particles (tegument and envelope) directly in the infected cells and the mTurquoise fluorescence image was used to identify the particles that were capsid positive.

Among the capsid-positive particles, two main types were observed: those containing both tegument and envelope (Capsid(+)/Tegument(+)/Envelope(+)) and others that were devoid of envelope (Capsid(+)/Tegument(+)/Envelope(−)) (). The particles exhibiting all viral components (Capsid (+)/Tegument(+)/Envelope(+)) displayed structures that were consistent with those observed in purified viruses (). There are several unique features to the M-cyclin–cdk2 structure; M-cyclin binds cdk2 with a relative disposition different from that of cyclin A such that structurally equivalent residues make different contacts with cdk2 with few interactions identical to the cyclin A–cdk2 interface (Figure C; Table ). We observed no clear visual difference in the distribution of VP1/2 between virus particles that were envelope positive and envelope negative. These data demonstrate that the use of two-colour dSTORM in combination with two-colour wide-field fluorescence (using genetically expressed mTurquoise-VP26 and gM-EYFP) provides super-resolution information about individual virions at different assembly steps. However, we noted that the mean localization precision (defined further on as 1 s.d.) achieved for the two-colour dSTORM images obtained both in cells and in purified viruses was of the order of 10–15 nm, irrespective of the fluorophore and the antibody used. We also observed that the fraction of localization with high-localization precision (5–10 nm) was much greater for AF647 than AF568, AF546 or ATTO532.

J. The order and orientation of the strands within the β-sheet is conserved: 3, 2, 1, 4, and 5, one of them being antiparallel to the other four (↑↓↑↑↑). However, it requires high spatial resolution and high labelling specificity, and typically involves complex data analysis. This complexity is notably due to the need for assessing effects of the localization error and the linker size between the protein of interest and the fluorescent label36,37. Here we present a structural analysis method taking these effects into account and apply it for the precise determination of the distribution of important tegument and envelope proteins in HSV-1. We acquired single-colour dSTORM images of purified viruses labelled with AF647. To establish why viral cyclins such as M- and K-cyclin form active complexes that are resistant to the cdk inhibitor p27Kip, we examined equivalent regions on cyclin A and M-cyclin that define the p27Kip-binding site.

We imaged a large number (>50) of particles and data sets were analysed as shown in (see also Supplementary Notes 1–3 and Supplementary Figs 1–3). We ensured that only fully assembled virus particles, for example, containing capsid, tegument and envelope, were used for the analysis. For this, capsid-positive particles were selected by creating a mask from the mTurquoise-VP26 fluorescence image. A second mask was also created from an additional wide-field fluorescence image using AF568 as described in the Methods section. Fontaine-Rodriguez, E. Asp651 is found at the beginning of α6, the last α-helix in the structure, which lies diagonally to the two β-strands on one of the faces of the central β-sheet (). By aligning the obtained particles, the localizations can be combined to reconstruct a high-density super-resolution image of a specific protein layer.

Furthermore, with the large number of localizations obtained in the aligned data set, the radial distribution of the aligned localizations can be accurately retrieved and analysed to determine the position of this individual layer. For this, the radial distribution of localizations was fitted to a Monte-Carlo model of virus localization data set (Monte-Carlo-based virus (MCV) model, presented in and Supplementary Note 2), developed assuming spherically symmetric virus particles. In this model, each viral protein is assumed to lie within a spherical shell described by two parameters: the shell diameter and the shell thickness. The helices of each cyclin are rendered as cylinders emphasizing the structural differences close to the M-cyclin N- … The shell diameter and thickness are the structural parameters of interest and are both obtained by fitting, whereas the linker size and localization precision are first estimated and then fixed during the analysis (Supplementary Note 3). The shell diameter represents the average radial position of the protein of interest in a population of virus particles. On the other hand, the shell thickness obtained by our method is a result of several contributions: the actual thickness of the protein layer (the thickness of the shell shown in ), the variability of the diameter from particle to particle in a population of virus particles and any deviation from the spherical symmetry.

You may also like