Live visualization of herpes simplex virus type 1 compartment dynamics

Abstract

We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.

FIG. 1.

Construction of rHSV-RYC. (A) Representation of the HSV-1 BAC (YEbac102) genome structure, showing the region containing the U_L35 gene. The galK expression cassette was inserted into the U_L35 gene through homologous recombination (HR) and selection for Gal⁺ recombinants (rHSVBAC35galK). GalK was then replaced by mRFP coding sequences by HR and counterselected for Gal⁻ recombinants (rHSVBAC35R). The same procedure was used for the fusion of EYFP with the U_L22 gene on the rHSVBAC35R genome (B) and for the fusion of ECFP with the U_L48 gene on rHSVBAC35R22Y (C). The BAC sequences were removed by the Cre/loxP recombination system (Cre), resulting in the recombinants HSV-1 rHSV-R, rHSV-RY, and rHSV-RYC. TR_L, terminal repeat of the long segment; U_L, unique long segment; IR_L, internal repeat of the long segment; IR_S, internal repeat of the short segment; U_S, unique short segment; TR_S, terminal repeat of the short segment.

FIG. 2.

Expression of fluorescent fusion proteins in infected cells. Vero cells were mock infected (m) or infected with either wt HSV-1 (wt), rHSV-RY (RY), rHSV-RYC (RYC), or rHSV-RYC/2 (RYC/2) at an MOI of 1 PFU and harvested when the CPE was approximately 90%. Cell lysates were analyzed by SDS-PAGE followed by Western blotting with antibodies against VP26 (A), VP16 (B), GFP (C), ICP4 (F), and ICP8 (G). For detection of gH and EYFP-gH, cell lysates were immunoprecipitated with a gH-specific antibody followed by SDS-PAGE and silver staining (D) or Western blotting with a GFP-specific antibody (E). Arrows indicate the mRFP-VP26, VP16-ECFP, and EYFP-gH fusion proteins, as well as the wt VP26, VP16, gH, ICP4, and ICP8 proteins. The arrowheads in panels D and E point to a band that likely represents the EYFP-gH precursor. Sizes of molecular weight standards are indicated.

FIG. 3.

Growth kinetics of recombinant HSV-1 and wt HSV-1. Vero cells were infected with MOIs of 0.1 (A and B) or 5 (C and D) PFU of either a recombinant HSV-1 (rHSV-R, rHSV-RY rHSV-RYC, rHSV-RC, or rHSV-48Y) or wt HSV-1, and progeny virus was harvested from the cell culture medium (A and C) or from the cells (B and D) at 0, 12, 24, 36, and 48 h p.i. Titers are expressed as PFU per ml. The titers represent means from three experiments. Error bars represent standard deviations.

FIG. 4.

Incorporation of fluorescent proteins into virions. Purified wt HSV-1 (wt), rHSV-RY (RY), rHSV-RYC (RYC), and rHSV-RYC/2 (RYC/2) were analyzed by SDS-PAGE followed by Western blot analysis with antibodies specific for VP26 (A), VP16 (B), GFP (C), and VP22 (F). For detection of gH and EYFP-gH, virion proteins were immunoprecipitated with a gH-specific antibody and analyzed by SDS-PAGE followed by silver staining (D) or Western blotting with a GFP-specific antibody (E). The corresponding fusion proteins mRFP-VP26, VP16-ECFP, and EYFP-gH, as well as the wt VP26, VP16, gH, and VP22 proteins are indicated. Unspecific bands are marked by asterisks. Sizes of molecular weight markers are shown.

FIG. 5.

High-resolution CLSM of living rHSV-RYC-infected cells. Vero cells were infected with an MOI of 18 PFU, and live cells were observed by CLSM with settings specific for ECFP (VP16-ECFP fusion protein), EYFP (EYFP-gH fusion protein), and mRFP (mRFP-VP26 fusion protein). The thin gray lines in panels a to d mark the contours of the nucleus. The insets in panels a to d show a magnification of the sector denoted by the white square. The filled arrowheads within the insets point to colocalizations of ECFP, EYFP, and mRFP signals. Arrows, early RCs; filled triangle, VP16-ECFP foci in periphery of RCs; open triangles, large mRFP-VP26 and VP16-ECFP foci in periphery of nuclei; filled diamonds, asymmetric, perinuclear accumulation of VP16-ECFP and mRFP-VP26. Images represent single z stacks of the cells.

FIG. 6.

High-resolution CLSM of rHSV-RYC-infected cells and rHSV-R- and rHSV-48Y-infected cells stained for ICP8. (A) Vero cells were infected with rHSV-RYC, and living cells were observed by CLSM as described for Fig. 5. The images show a high magnification of a protrusion of the plasma membrane (top of picture). The arrows point to foci in which all three fusion proteins colocalize. The insets show magnifications of these foci. (B) Vero cells were infected with rHSV-RYC, and fixed cells were observed by CLSM as described for panel A. (C) Vero cells were infected with rHSV-R at an MOI of 10 PFU, fixed at 12 h p.i., and stained with the anti-ICP8 MAb 7381 and a FITC-conjugated secondary antibody, as well as DAPI. The cells were observed by CLSM with settings specific for DAPI, FITC (ICP8), and mRFP (mRFP-VP26 fusion protein). (D) Vero cells were infected with rHSV-48Y at an MOI of 10 PFU and stained as described for panel C, except that an AF594-conjugated secondary antibody was used. The cells were observed by CLSM with settings specific for DAPI, EYFP (VP16-EYFP fusion protein), and AF594 (ICP8). Images in panels A to D represent single z stacks of the cells.

FIG. 7.

High-resolution CLSM of large VP26 foci. (A) Vero cells were infected with rHSV-RY at an MOI of 0.3 PFU, fixed at the indicated times postinfection, stained with DAPI, and observed by CLSM with settings specific for DAPI, EYFP (EYFP-gH fusion protein), and mRFP (mRFP-VP26 fusion protein). Images represent single z stacks of the cells. (B) Panels a and c show magnifications of the mRFP-VP26 foci marked with the numbered arrows shown in panel A, while panels b and d show surpass views of three-dimensional reconstructions of the same foci. (C) Vero cells were infected with wt HSV-1 at an MOI of 10 PFU, fixed at 12 h p.i., and stained with the rabbit anti-VP26 PAb VP26/C and a FITC-conjugated secondary antibody. HSV-1 DNA was detected by FISH using an Atto590-labeled probe. Cells were observed by CLSM with settings specific for DAPI, FITC (VP26), and Atto590 (HSV-1 DNA). Images represent single z stacks of the cells.

FIG. 8.

Immunofluorescence staining for VP16, gH, and VP26 in wt HSV-1-infected cells. (A) Immunofluorescence staining for VP16. Vero cells were infected with wt HSV-1 at an MOI of 10 PFU. At the indicated times postinfection, the cells were fixed and stained with the anti-VP16 MAb LP1 and an AF594-conjugated secondary antibody, as well as DAPI. The cells were then observed by CLSM with settings specific for DAPI and AF594. Filled arrowhead, VP16 foci in the periphery of RCs. (B) Immunofluorescence staining for gH. Vero cells were infected and stained as described for panel A, except that the anti-gH MAb LP11 was used. (C) Immunofluorescence staining for VP26. Vero cells were infected and stained as described for panel A, except that the rabbit anti-VP26 PAb VP26/C was used. Panel h shows a magnification of the VP26 focus marked with an arrow. (D) Vero cells were infected as described for panel A, fixed at 16 h p.i., and stained with DAPI, the anti-VP16 MAb LP1 (detected with an AF594-conjugated secondary antibody), and the rabbit anti-VP26 PAb VP26/C (detected with a FITC-conjugated secondary antibody). The cells were then observed by CLSM with settings specific for DAPI, AF594 (VP16, shown in green), and FITC (VP26, shown in red). Images in panels A to D represent single z stacks of the cells.

FIG. 9.

Time-lapse CLSM of rHSV-RYC- and rHSV-RY-infected cells. Vero cells were infected with rHSV-RYC (A) or rHSV-RY (B) at MOIs of 2 and 0.3, respectively. Cells that just started to accumulate fluorescent proteins were monitored by CLSM with settings specific for ECFP (VP16-ECFP), EYFP (EYFP-gH), and mRFP (mRFP-VP26). Selected frames at the indicated intervals are shown. Images were processed with Imaris software in the surpass view mode. Arrowheads denote the cells described in the text.

FIG. 10.

Electron micrographs of Vero cells infected with rHSV-RYC after prefixation at 24 h p.i. followed by freezing and freeze-substitution. (A) Low-power micrograph showing Golgi membranes, virions within a vacuole (arrow), and capsids (arrowheads) within the cytoplasm and budding at Golgi membranes, respectively. (B) Virion within the perinuclear space of the nuclear envelope. (C) Accumulation of capsids in a crystalline manner within the nucleus. (D) Budding capsid at Golgi membranes. (E) Virion within a concentric vacuole. (F) Virions in the extracellular space. Bars, 500 nm (A and C) and 100 nm (B, D, and E).