The pseudorabies virus VP22 homologue (UL49) is dispensable for virus growth in vitro and has no effect on virulence and neuronal spread in rodents

Abstract

The tegument of herpesvirus virions is a distinctive structure whose assembly and function are not well understood. The herpes simplex virus type 1 VP22 tegument protein encoded by the UL49 gene is conserved among the alphaherpesviruses. Using cell biology and viral genetics, we provide an initial characterization of the pseudorabies virus (PRV) VP22 homologue. We identified three isoforms of VP22 present in PRV-infected cells that can be resolved by polyacrylamide gel electrophoresis. The predominant form is not phosphorylated and is present in virions, while the other two species are phosphorylated and excluded from virions. VP22 localized to the nucleus by 6 h postinfection, as determined by immunofluorescence and cell fractionation. VP22 immunofluorescence in the nucleus was both diffuse and in punctate structures. The punctate nuclear localization was the most pronounced form of staining and did not localize exclusively to sites of viral DNA replication. Unexpectedly, a VP22 null mutant had no obvious phenotypes during tissue culture infections and was similar to the wild type in all respects. Moreover, the VP22 null mutant was as virulent and neuroinvasive as the wild-type virus after infection of the rodent eye and spread to the brain using both anterograde and retrograde neuronal circuits.

FIG. 1.

PRV 175 is a VP22-null virus which expresses GFP. (A) Shown is a BamHI/SalI fragment of PRV Becker (Be) containing the UL49 gene. PRV 175 is identical to Becker except that the XhoI/PstI fragment of UL49 has been replaced with the EGFP gene. (B) Monolayers of PK15 cells were either mock infected or infected with PRV Becker (wild type), 175 (VP22-null), or 175R (revertant of PRV 175) at an MOI of 10 for 16 h prior to preparation of whole-cell lysates. The same infected cell lysates were separated on two polyacrylamide gels, and Western blot analysis was performed with either rabbit polyclonal antiserum to VP22 (top) or polyclonal antiserum to EGFP (bottom). The migration positions of molecular mass markers are shown on the left (in kilodaltons).

FIG. 2.

Compartmentalization of VP22 in infected cells and purified virions. Monolayers of PK15 cells were infected at an MOI of 10 for the times indicated (16 or 6 h) prior to preparation of cell lysates. Cells were either mock infected (M) or infected with PRV Becker from which total cell lysate (T), cytoplasmic (C), or nuclear (N) fractions were isolated. Purified extracellular virions (V) were treated with either 1% NP-40 or 1% NP-40 plus 1 M NaCl, from which soluble (S) and pellet (P) fractions were isolated by high-speed centrifugation. Western blot analysis was performed with either rabbit polyclonal antiserum to VP22 (top) or goat polyclonal antiserum to gC (bottom). The two forms present at 16 h postinfection (hpi) are indicated with asterisks. The migration positions of molecular mass markers are shown on the left (in kilodaltons).

FIG. 3.

Subcellular localization of VP22 by indirect immunofluorescence. PK15 cells grown on cover slips were mock infected (A) or infected at an MOI of 10 with PRV Becker (B and C) for 6 h prior to fixation. Cells were stained with rabbit polyclonal antiserum to VP22 and detected with an Alexa-488 secondary antibody. Magnifications: ×120 (A and B) and ×160 (C).

FIG. 4.

Localization of VP22 and sites of viral replication. CV-1 cells grown on cover slips were incubated in reduced serum and 3 mM n-butyrate for 16 h prior to infection. The cells were infected in reduced serum and 3 mM n-butyrate at an MOI of 10 with PRV Becker. At 6 h postinfection, BrdU was added to the medium for 15 min (A to D) or 60 min (E to H) prior to fixation. Cells were then stained with polyclonal antiserum to VP22, fixed again, treated with 4 N HCl, and stained with monoclonal antiserum to BrdU. Detection of BrdU was performed with an Alexa-546 secondary antibody and is shown in red (B and F), while VP22, shown in green (A and E), was detected with an Alexa-488 secondary antibody. Merged BrdU and VP22 staining is shown in C and G). A region of costaining (C and G, indicated by the inset) is shown under higher magnification (D and H). Magnifications: ×160 (A to C, E to G) and ×480 (D and H).

FIG. 5.

Radioimmunoprecipitation assay of VP22 in infected cells. Monolayers of PK15 cells were infected at an MOI of 10 with PRV Becker or 175 and labeled overnight in the presence of either [³⁵S]methionine-cysteine or [³³P]orthophosphate. Total cellular extracts (lanes 1 to 4) or purified wild-type extracellular virions (lanes 5 and 6) were prepared at 16 h postinfection and subjected to immunoprecipitation with rabbit polyclonal antiserum to VP22. Upon immunoprecipitation, one [³⁵S]methionine-cysteine-labeled Becker sample was treated with lambda protein phosphatase (PPase) (lane 3). The immunoprecipitated products were separated by SDS-12.5% PAGE and visualized by autoradiography. The three observed electrophoretic forms of VP22 are indicated on the left (a to c). The migration positions of molecular mass markers are shown on the right (in kilodaltons).

FIG. 6.

Anterograde and retrograde spread of PRV 175 in the rodent visual system. Approximately 5 × 10⁵ PFU of PRV Becker (Be) or 175 was injected into the vitreous humor of Sprague-Dawley male rats. At the time of death, the brains were fixed and sliced into 35-μm-thick coronal sections with a freezing microtome. Viral antigen was detected with polyvalent antiserum Rb133. Representative sections containing suprachiasmatic nuclei (SCN), LGN including the dorsal (D) and ventral (V) aspects, as well as the intergeniculate leaflet (IGL), superior colliculus (SC), and the Edinger-Westphal nucleus (EW) are pictured. The infection of the ipsilateral EW nucleus by PRV Becker and of both nuclei by PRV 175 reflects the kinetics of multisynaptic spread from the pretectal nucleus across the posterior commissure to the contralateral EW nucleus. Intensity of infection of the contralateral nucleus varied from animal to animal.