The herpes simplex virus 1 UL51 protein interacts with the UL7 protein and plays a role in its recruitment into the virion

Abstract

The alphaherpesvirus UL51 protein is a tegument component that interacts with the viral glycoprotein E and functions at multiple steps in virus assembly and spread in epithelial cells. We show here that pUL51 forms a complex in infected cells with another conserved tegument protein, pUL7. This complex can form in the absence of other viral proteins and is largely responsible for recruitment of pUL7 to cytoplasmic membranes and into the virion tegument. Incomplete colocalization of pUL51 and pUL7 in infected cells, however, suggests that a significant fraction of the population of each protein is not complexed with the other and that they may accomplish independent functions.

Importance: The ability of herpesviruses to spread from cell to cell in the face of an immune response is critical for disease and shedding following reactivation from latency. Cell-to-cell spread is a conserved ability of herpesviruses, and the identification of conserved viral genes that mediate this process will aid in the design of attenuated vaccines and of novel therapeutics. The conserved UL51 gene of herpes simplex virus 1 plays important roles in cell-to-cell spread and in virus assembly in the cytoplasm, both of which likely depend on specific interactions with other viral and cellular proteins. Here we identify one of those interactions with the product of another conserved herpesvirus gene, UL7, and show that formation of this complex mediates recruitment of UL7 to membranes and to the virion.

FIG 1

Construction of recombinant viruses. (A) Schematic diagram of the HSV-1(F) genome (line 1) and of the recombinant viruses used in this study. TRL, terminal repeat long region; UL, unique long region; IRL, internal repeat long region; IRS, internal repeat short region; US, unique short region; TRS, terminal repeat short region. Line 2, structures of the wild-type sequences in the regions of UL51 and US8; line 3, the UL51Δ73-244 virus carries a stop codon and a kanamycin resistance cassette in place of the sequences coding for amino acids 73 to 244 of pUL51; line 4, the UL51-FLAG virus carries a FLAG tag at the C terminus of UL51 followed by a kanamycin resistance cassette; line 5, the UL51Δ167-244FLAG virus was constructed as described in Materials and Methods and carries a FLAG-tagged truncated pUL51 that lacks approximately the last third of the protein. (B) UL51 sequence conservation. The plot shows the conservation of the biochemical properties of amino acids using all available herpesvirus pUL51 homologous sequences aligned using the program MUSCLE (87). Each residue position receives a conservation score, and scores were averaged over a sliding 5-amino-acid-residue window.

FIG 2

Copurification and coimmunoprecipitation of pUL7 with pUL51. (A) Coomassie brilliant blue-stained SDS-polyacrylamide gel containing proteins purified from infected HEp-2 cells using anti-FLAG magnetic beads. White arrowheads, FLAG-tagged pUL51 proteins; small black arrowheads, copurifying proteins that partially comigrated with full-length pUL51. Numbers on the left are molecular sizes (in kilodaltons). (B) Immunoblot of proteins from Vero or HEp-2 cells infected with the viruses indicated below each lane. (Top) pUL7 present in the lysate from infected cells; (bottom) pUL7 present in eluents from anti-FLAG purification. (C) Immunoblot of proteins from HEp-2 cells infected with wild-type virus. The leftmost lane contains proteins from cell lysates. The immunoprecipitating (IP) antibody is indicated above each lane, and the antibody used for probing of the blot is indicated to the left of each panel.

FIG 3

Localization of pUL51, pUL7, and gE in infected cells. Vero cells infected for 14 h with HSV-1 UL51WT-FLAG (A to D) or HSV-1 UL51Δ73-244 (E to G) were fixed and probed with anti-FLAG antibody to detect pUL51 (A and D), anti-UL7 antiserum (B, D, E, and G), and mouse monoclonal antibody directed against gE (C, D, F, and G). Confocal images of single z-sections taken near the center of the nucleus (i.e., where the nuclear cross-section is the largest) with a 60× oil objective are shown. Negative controls (not shown) were normal rabbit serum for anti-UL7 rabbit antiserum and uninfected cells for anti-gE and anti-FLAG and showed no fluorescence at the laser and detector settings used to obtain these images. (H) Quantitation of gE and pUL7 colocalization. gE and pUL7 fluorescent intensities were measured across the linear profiles of 10 randomly selected cells, as described in Materials and Methods. Representative profiles from one cell infected with UL51-FLAG (top) and UL51Δ73-244 (bottom) are shown along with the profile plots and derived Pearson correlation coefficients. Aggregate results from 10 profiles are shown at the right.

FIG 4

Localization of pUL51, pUL7, and gE in transfected cells. Vero cells were transfected with plasmids for 24 h and then fixed and probed by indirect immunofluorescence. Images of single z-sections taken near the center of the nucleus with a 60× oil objective are shown. Primary antibodies are indicated at the tops of the panels, and the plasmids used for transfection are indicated to the left of each row. White arrowheads, areas of colocalization between pUL51 and pUL7.

FIG 5

Recruitment of pUL7 to virions by pUL51. (A) Immunoblots of virion gradient fractions from fractionation of cytoplasmic membranes of cells infected with wild-type or UL51Δ73-244 virus are shown. (B) Immunoblots of virion gradient fractions from fractionation cell culture supernatants of cells infected with wild-type or UL51Δ73-244 virus are shown. For panels A and B, the infecting virus is indicated to the left of each panel. The antibody used for immunoblot probing is indicated to the right. (C) Plot of infectivity in the gradients shown in panel B.