Selection of HSV capsids for envelopment involves interaction between capsid surface components pUL31, pUL17, and pUL25

Abstract

During egress from the nucleus, HSV capsids that contain DNA (termed C capsids) are preferentially enveloped at the inner nuclear membrane over capsid types lacking DNA. Using coimmunoprecipitation and biochemical analyses of wild-type and mutant capsids, we identify an interaction between a complex of pU(L)17/pU(L)25, termed the C capsid-specific complex (CCSC), and pU(L)31, a component of the nuclear egress complex (NEC). We also show that the interactions between these components are dependent on expression of all three proteins but occur independently of the pU(L)31 interacting protein and NEC component pU(L)34, as well as a kinase encoded by U(S)3 that phosphorylates both pU(L)31 and pU(L)34. The interaction between the CCSC and pU(L)31 in the NEC suggests a mechanism to conserve viral resources by promoting assembly of only those viral particles with the potential to become infectious.

Fig. 1.

Coimmunoprecipitation of pU_L17, pU_L25, and pU_L31 requires expression of all three proteins. CV1 cells were infected with HSV-1(F), U_L17 null, U_L25 null, or U_L31 null viruses, or were mock infected. At 18 h after infection cells were lysed, and soluble lysates (Left) or immunoprecipitation reactions (Right) using anti-pU_L17 or anti-pU_L31 antibodies were electrophoretically separated and immunoblotted with anti-pU_L25, anti-U_L31, anti-pU_L17, or anti-VP16 antibodies.

Fig. 2.

Immunoblot of sucrose gradient-fractionated wild-type capsids probed with anti-pU_L31, anti-pU_L25, or anti-VP5 or anti-pU_L17 antibodies. CV1 cells were infected with HSV-1(F) at a multiplicity of infection of 5 pfu/cell. At 20 h after infection cells were collected and lysed. Capsids were pelleted by centrifugation through a sucrose cushion and were then resuspended and separated on a continuous sucrose gradient (Materials and Methods). Approximately 0.5-mL fractions as determined by eye were collected from the bottom of the gradient (fraction 1) to the top (fraction 20) using a Buchler Auto Bensi-Flow IIC gradient collector. Proteins in fractions were TCA precipitated, and pellets were denatured and solubilized in SDS. Fractions 2 through 19 were separated on an SDS polyacrylamide gel and analyzed by immunoblotting, followed by reaction with appropriate conjugates, application of chemiluminescence substrate, exposure to X-ray film, and digital scanning. (A) Top and Upper Middle: Images of the same blot first probed with pU_L31 then stripped and reprobed with VP5 antibodies. Lower Middle and Bottom: A second blot containing identical samples probed with pU_L25 antibody. Immunoreactivity was then stripped, and the blot was probed with pU_L17 antibody. (B) Percentage of immunoreactivity of a given antibody in different fractions as quantified by Image J software.

Fig. 3.

pU_L31 capsid association requires pU_L25 but not pU_L17 nor pU_L34. CV1 cells were infected with 5.0 pfu/cell of (A) U_L25 null, (B) U_L17 null, or (C) U_L34 null viruses. Cellular lysates prepared at 20 h after infection were pelleted through a sucrose cushion. Material in the pellets was resuspended and separated by ultracentrifugation through 10 mL continuous sucrose gradients. Twenty ≈0.5-mL fractions were collected from bottom to top of each gradient. Material in fractions was TCA precipitated and solubilized in SDS. Fractions 2–19 (A and B) from the U_L25 null and U_L17 null gradients and fractions 2–18 (C) of the U_L34 null gradient were denatured in SDS, electrophoretically separated, and subjected to immunoblotting with VP5 and pU_L31 specific antibodies.

Fig. 4.

The pU_L17/pU_L25/pU_L31 complex forms in the absence of pU_L18, intact capsids, pU_L34, or pU_S3. CV1 cells were infected with HSV-1(F), U_L18 null, U_L34 null, or U_S3 null viruses. At 18 h after infection cells were lysed and immunoprecipitated with anti-pU_L17 (Left) or anti-pU_L31 (Right) antibodies. The immunoprecipitated material was then subjected to immunoblotting with anti-pU_L25, anti-U_L31, or anti-pU_L17 antibodies.

Fig. 5.

Model of nuclear capsid egress. Icosahedral C capsids contain DNA, B capsids lack DNA but contain an internal proteinaceous scaffold (black circle), and A capsids lack internal contents. The nuclear lamina is perforated by the action of cellular and viral kinases (such as viral U_S3) to allow nucleocapsids access to the INM. pU_L17 and pU_L25 interact with one another and with pU_L31 in the nucleoplasm. The pU_L17/pU_L25/pU_L31 complex then attaches to capsids (step 1). This complex is present on all capsid types but enriched on the surface of C capsids. The pU_L31 moiety in this complex then interacts with pU_L34 at the INM or a complete pU_L31/pU_L34 complex (the nuclear envelopment complex or NEC; step 2). The larger number of pU_L17/pU_L25/pU_L31 complexes on C capsids may recruit locally high concentrations of NECs to trigger envelopment (step 3). During envelopment, the viral kinase pU_S3 is also incorporated into the perinuclear virion. Deenvelopment (step 4) is triggered by pU_S3-mediated phosphorylation of pU_L31 and viral glycoprotein gB (gB is not diagrammed). pU_L31 is retained in association with pU_L34 at the outer nuclear membrane, whereas the capsid is released into the cytosol for eventual budding at cytoplasmic membranes.