Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE

Abstract

The role of alphaherpesvirus membrane protein internalization during the course of viral infection remains a matter of speculation. To determine the role of internalization of the pseudorabies virus (PRV) gE and gI proteins, we constructed viral mutants encoding specific mutations in the cytoplasmic tail of the gE gene that inhibited internalization of the gE-gI complex. We used these mutants to assess the role of gE-gI endocytosis in incorporation of the proteins into the viral envelope and in gE-mediated spread or gE-promoted virulence. In addition, we report that another viral mutant, PRV 25, which encodes a gE protein defective in endocytosis, contains an additional, previously uncharacterized mutation in the gE gene. We compared PRV 25 to another viral mutant, PRV 107, that does not express the cytoplasmic tail of the gE protein. The gE protein encoded by PRV 107 is also defective in endocytosis. We conclude that efficient endocytosis of gE is not required for gE incorporation into virions, gE-mediated virulence, or spread of virus in the rat central nervous system. However, we do correlate the defect in endocytosis to a small-plaque phenotype in cultured cells.

FIG. 1

Amino acid comparison of gE proteins. The amino acid sequences of the transmembrane domains and cytoplasmic tails of gE proteins encoded by PRV Be (wild-type), PRV 25 (frameshift), and PRV 107 (Am457) are shown. The transmembrane domain of each protein is underlined. Potential internalization motifs, either tyrosine or dileucine based, are in shown in bold in the wild-type sequence. The two acidic amino acid clusters are shown in italics. An asterisk indicates a stop codon in translation of the protein. PRV 25 contains a shift in reading frame predicted to result in a truncated transmembrane domain and a novel cytoplasmic tail. PRV 107 contains an amber stop codon in place of an arginine residue just after the transmembrane domain and encodes a truncated, anchored gE protein.

FIG. 2

Expression of gE proteins. PK15 cells were infected at an MOI of 10 with either PRV Be (wild type), PRV 107 (Am457), PRV 104 (Y478S), PRV 105 (Y478S + Y517S), PRV 106 (Y517S), PRV 107R (revertant PRV 107), or PRV 25 (frameshift) for 16 h prior to preparation of cell lysates. Western blot analysis was performed with either rabbit polyclonal antiserum to gE (A) or goat polyclonal antiserum to gC (B). In lanes 1 to 7 of panel A, 10 μg of total protein was loaded in each lane; 60 μg of total protein was loaded in lane 8. Immature wild-type gE protein has a molecular mass of approximately 93 kDa and a mature form of approximately 110 kDa. Cells infected with PRV 107 contain immature and mature forms of approximately 70 and 93 kDa, respectively. The gE protein produced by PRV 25 has an immature form of 80 kDa and a mature form of 101 kDa. In panel B, 10 μg of total protein was analyzed for each sample. Positions of apparent molecular mass markers are shown on the left in kilodaltons.

FIG. 3

Steady-state distribution of the gE-gI complex. PK15 and MDBK cells were infected at an MOI of 10 for the times indicated with either PRV Be (wild type), PRV 25 (frameshift), PRV 107 (Am457), PRV 104 (Y478S), PRV 105 (Y478S + Y517S), or PRV 106 (Y517S). The cells were fixed, permeabilized, and reacted with a MAb that specifically recognized gE when it was complexed with gI (MAb 1/14). An Alexa-568-conjugated secondary antibody was used to visualize bound MAb. Confocal sections were taken through the centers of the cells.

FIG. 4

Endocytosis of the gE mutant-gI complex. PK15 (A) and MDBK (B) cells were infected at an MOI of 10 with either PRV Be (wild type), PRV 107 (Am457), PRV 104 (Y478S), PRV 105 (Y478S + Y517S), or PRV 106 (Y517S) for 4 h prior to an indirect immunofluorescence endocytosis assay as described in Materials and Methods. Briefly, the cells were incubated at 4°C with MAb 1/14, which specifically recognized gE when it was complexed with gI prior to a shift of the cells to 37°C for the indicated times to allow internalization of the protein complex. The cells were then fixed, permeabilized, and reacted with an Alexa-568-conjugated secondary antibody to visualize the bound primary antibody. Confocal sections were taken through the centers of the cells.

FIG. 4

Endocytosis of the gE mutant-gI complex. PK15 (A) and MDBK (B) cells were infected at an MOI of 10 with either PRV Be (wild type), PRV 107 (Am457), PRV 104 (Y478S), PRV 105 (Y478S + Y517S), or PRV 106 (Y517S) for 4 h prior to an indirect immunofluorescence endocytosis assay as described in Materials and Methods. Briefly, the cells were incubated at 4°C with MAb 1/14, which specifically recognized gE when it was complexed with gI prior to a shift of the cells to 37°C for the indicated times to allow internalization of the protein complex. The cells were then fixed, permeabilized, and reacted with an Alexa-568-conjugated secondary antibody to visualize the bound primary antibody. Confocal sections were taken through the centers of the cells.

FIG. 5

Incorporation of gE proteins into virions. PK15 cells were infected at an MOI of 10 with either PRV Be (wild type), PRV 107 (Am457), PRV 25 (frameshift), PRV 104 (Y478S), PRV 105 (Y478S + Y517S), or PRV 106 (Y517S) for 14 h. Medium was removed from the cells, and released virions were isolated by centrifugation through 30% sucrose. Western blot analysis was performed with a rabbit polyclonal antiserum to gE. The same total volume of extract was loaded in each lane except for lane 3, in which twice as much total protein was analyzed. Positions of apparent molecular mass markers are indicated in kilodaltons on the left.

FIG. 6

Localization of viral antigen in brain sections. The brains from animals taken from infections described in the legend to Fig. 7 were removed and analyzed for viral antigen with a polyvalent rabbit antiserum generated against whole virus particles (Rb133). Serial sections (35 μm) through the coronal plane were cut, processed and mounted on slides. Representative sections containing the SCN, LGN including the dorsal (D) and ventral (V) aspects as well as the IGL, and the SC are pictured.

FIG. 7

Distribution of time to symptoms for each infected animal. Animals were infected with either PRV Be (wild type), PRV 104 (Y478S), PRV 105 (Y478S + Y517S), PRV 106 (Y517S), or PRV 107 (Am457) by intraocular injection as described in Materials and Methods. The time at which signs of imminent death were evident for each animal was noted before the animals were sacrificed.