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. 1999 Aug;73(8):6769-81.
doi: 10.1128/JVI.73.8.6769-6781.1999.

Modified VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection

L E Pomeranz  1 , J A Blaho
Affiliations

Modified VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection

L E Pomeranz et al. J Virol. 1999 Aug.

Abstract

The UL49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was "undermodified" in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [32P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection.

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Figures

FIG. 1
FIG. 1
Indirect immunofluorescence of HSV-1(F)-infected cell plaques singly labeled with antibodies specific for VP22 (A and C) and ICP22 (B) and doubly labeled with antibodies specific for VP22 and ICP4 (D to I). Vero cells were infected at an MOI of 0.001 with HSV-1(F) for 51 h, fixed with formaldehyde, permeabilized with acetone, and stained with antibodies RGST49 (A and C), RGST22 (B), affinity-purified RGST49 (D, F, G, and I), or 1114 (E, F, H, and I) as described in Materials and Methods. Abbreviations: un, uninfected cells; n, c, and d nuclear, cytoplasmic, and diffuse subcellular localizations of VP22, respectively. Low magnifications were ×10 (D to F) and ×20 (A and B); high magnifications were ×40 (G to I) and ×63 (C).
FIG. 2
FIG. 2
Phase-contrast (D) and indirect immunofluorescence (A to C and E to G) of HSV-1(F) plaques doubly labeled with antibodies specific for VP22 and ICP4. Vero cells were infected at an MOI of 0.001 with HSV-1(F) for 51 h in parallel with those in Fig. 1D to I, fixed and permeabilized with 100% methanol (17), and stained with affinity-purified RGST49 (A, C, E, and G) and 1114 (B, C, F, and G) as described in Materials and Methods.
FIG. 3
FIG. 3
Indirect immunofluorescence of HSV-1(F)-infected cells doubly labeled with antibodies specific for VP22 (B, E, H, and K) and ICP4 (A, D, G, and J). Vero cells were infected with 0.1 PFU of HSV-1(F) per ml on ice at 4°C for 1 h. 1 h p.i. refers to the point at which fresh (37°C) medium was added back to the flasks. Infections were terminated at 7, 9, 13, and 25 h p.i. prior to formaldehyde-acetone fixation followed by immunostaining with affinity-purified RGST49 and 1101 as described in Materials and Methods, n, c, and d refer to the nuclear, cytoplasmic, and diffuse subcellular localizations of VP22, respectively (Fig. 1). Overlays are shown in panels C, F, I, and L.
FIG. 4
FIG. 4
Double-label indirect immunofluorescence of synchronously infected cells stained with antibodies specific for VP22 (B, E, H, and K) and gD (A, D, G, and J). Vero cells were synchronously infected with HSV-1(F) (MOI = 30) and at 5, 7, 9, 13 h p.i. were formaldehyde fixed and acetone permeabilized before being immunostained with affinity-purified RGST49 and 1103 as described in Materials and Methods. 1 h p.i. refers to the point at which fresh (37°C) medium was added back to the flasks following a 1-h absorption at 4°C. Overlays are shown in panels C, F, I, and L.
FIG. 5
FIG. 5
Confocal indirect immunofluorescence of synchronously infected cells stained with antibodies specific for VP22. Vero cells were synchronously infected with HSV-1(F) (MOI = 30) or mock infected and at 3, 5, 7, 9, 13 h p.i. were formaldehyde fixed and acetone permeabilized before being immunostained with affinity-purified RGST49 as described in Materials and Methods. Confocal images were obtained with a Leica laser-scanning microscope. The mock control at 13 h p.i. is shown. 1 h p.i. refers to the point at which fresh (37°C) medium was added to the flasks following a 1-h adsorption at 4°C.
FIG. 6
FIG. 6
Immunoreactivities of infected cell proteins extracted during the course of a synchronized HSV-1(F) infection. Vero cells were synchronously infected with HSV-1(F) (MOI = 30) or mock infected. At 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 h p.i., whole-cell extracts were prepared and polypeptides were separated in a denaturing gel and transferred to nitrocellulose prior to chemiluminescent staining with antibodies RGST22 and RGST49 as described in Materials and Methods. The dot marks the slowest-migrating form of VP22, and the vertical line marks the faster form(s). 1 h p.i. refers to the point at which fresh (37°C) medium was added back to the flasks following a 1-h absorption at 4°C.
FIG. 7
FIG. 7
Immunoreactivities of polypeptides derived from either whole-cell extracts (WCE) or nuclear (Nuc) and cytoplasmic (Cyto) fractions during the course of a synchronized HSV-1(F) infection. Vero cells were synchronously infected with HSV-1(F) (MOI = 30) or mock infected. At 1, 5, 9, and 13 h p.i., either whole-cell extracts or nuclear and cytoplasmic fractions were prepared and polypeptides were separated in a denaturing gel and transferred to nitrocellulose before undergoing chemiluminescent staining with antibody G49 as described in Materials and Methods. Dots mark the locations of the three observed electrophoretic forms of VP22 (a, b, and c). 1 h p.i. refers to the point at which fresh (37°C) medium was added back to the flasks following a 1-h adsorption at 4°C. The location of the 45-kDa molecular mass marker is indicated (MW).
FIG. 8
FIG. 8
Autoradiographic images (A) and immunoreactivities (B) of 32P-orthophosphate-labeled infected-cell polypeptides immunoprecipitated with an anti-VP22 antibody during the course of a synchronized HSV-1(F) infection. Vero cells were synchronously infected with HSV-1(F) (MOI = 30) or mock infected in radiolabel-free medium. At 5 h p.i., medium containing 32P-orthophosphate was added, and after a 2-h incubation, radiolabel was removed. At 9 and 13 h p.i., whole-cell extracts (WCE), nuclear (N) and cytoplasmic (C) fractions, or the residual matrices were precipitated with the RGST49 antibody as described in Materials and Methods. Immunoprecipitated (IP) infected-cell polypeptides were separated in a denaturing 15% DATD-acrylamide gel, transferred to nitrocellulose, and reacted with antibody RGST49. An alkaline phosphatase-conjugated secondary antibody was used for immunostaining prior to autoradiography. The solid arrows mark the location of VP22, while the open arrowheads mark the potential degradation product referred to in the text.
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