The capsid and tegument of the alphaherpesviruses are linked by an interaction between the UL25 and VP1/2 proteins

Abstract

How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.

FIG. 1.

The VP1/2 CBD is conserved in HSV-1 and PRV. (A) Vero cells transiently expressing VP1/2cbd from either HSV-1 or PRV fused to the mCherry fluorescent protein. (B) mCherry-VP1/2cbd-expressing cells infected with HSV-1 or PRV encoding GFP-capsid fusions and imaged at 6 to 8 hpi. DIC, differential interference contrast.

FIG. 2.

UL25 is required for VP1/2cbd localization to capsid assemblons. Vero cells were transfected with the PRV mCherry-VP1/2cbd construct and subsequently infected with PRV encoding GFP-capsid fusions and either ΔUL36, ΔUL25, or a revertant of ΔUL25 (UL25R). Cells were imaged at 6 to 8 hpi. DIC, differential interference contrast.

FIG. 3.

VP1/2cbd is associated with capsids isolated from PRV-infected cell nuclei. (A) Representative image of capsids isolated from the infected cell nuclei. (B) Fluorescence imaging of capsids isolated from PK15 cells stably expressing GFP-VP1/2cbd infected with wild-type RFP-capsid virus (left panels), ΔUL25 RFP-capsid virus (center panels), or PK15 cells infected with an RFP-capsid/GFP-VP22 virus (right panels). Arrowheads indicate capsids emitting detectable GFP fluorescence. (C) Percentage of RFP-capsid fusions emitting detectable GFP fluorescence, as shown in panel B. Error bars are standard deviations. (D) The presence of VP5 and GFP fusion proteins was detected in each capsid preparation by Western blot analysis. Predicted molecular sizes of proteins are indicated at right, which were consistent with molecular size markers. α, anti.

FIG. 4.

VP1/2cbd and UL25 interact in the absence of other viral proteins. (A) Illustration of GFP-VP1/2 fusion proteins. (B and D) PRV UL25-myc detection by Western blotting either directly from cell lysates or after immunoprecipitation from lysates with an anti-GFP antibody. HEK-293 cells were cotransfected with PRV UL25-myc and either PRV GFP-VP1/2 (FL, full-length), GFP-VP1/2Δcbd (Trunc, truncated), PRV GFP-VP1/2cbd (CBD), unfused GFP, or no additional construct. (C) Densitometry analysis of three duplicate experiments as documented in panel B. For each sample, the ratio of the amount of UL25-myc in the immunoprecipitate to the amount in the lysate is shown relative to the background (no GFP sample). Error bars are standard deviations. (E) HSV-1 VP1/2cbd detection by Western blotting from HEK-293 cells cotransfected with or without an HSV-1 UL25-mCherry fusion construct. Predicted molecular sizes of proteins are indicated at left, which were consistent with molecular size markers. α, anti; WB, Western blotting; IP, immunoprecipitation.

FIG. 5.

UL25 does not require VP1/2 to localize to capsid assemblons. Vero cells were transfected with a PRV mCherry-UL25 construct and subsequently infected with wild-type, ΔUL25, or ΔUL36 PRV encoding GFP-capsid fusions. Cells were imaged at 6 to 8 hpi.

FIG. 6.

Illustration of capsid and tegument protein interactions. Protein interactions are based on the findings of this study and previous reports (27, 59). The juxtaposition of the protein interactions is based on the findings presented in this report that indicate that the VP1/2-binding proteins, UL37 and VP16, do not participate in the newly described VP1/2-UL25 interaction. N, amino terminus; C, carboxy terminus.