Herpes Simplex Virus 1 Recruits CD98 Heavy Chain and β1 Integrin to the Nuclear Membrane for Viral De-Envelopment

Abstract

Herpesviruses have evolved a unique mechanism for nucleocytoplasmic transport of nascent nucleocapsids: the nucleocapsids bud through the inner nuclear membrane (INM; primary envelopment), and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Little is known about the molecular mechanism of herpesviral de-envelopment. We show here that the knockdown of both CD98 heavy chain (CD98hc) and its binding partner β1 integrin induced membranous structures containing enveloped herpes simplex virus 1 (HSV-1) virions that are invaginations of the INM into the nucleoplasm and induced aberrant accumulation of enveloped virions in the perinuclear space and in the invagination structures. These effects were similar to those of the previously reported mutation(s) in HSV-1 proteins gB, gH, UL31, and/or Us3, which were shown here to form a complex(es) with CD98hc in HSV-1-infected cells. These results suggested that cellular proteins CD98hc and β1 integrin synergistically or independently regulated HSV-1 de-envelopment, probably by interacting directly and/or indirectly with these HSV-1 proteins.

Importance: Certain cellular and viral macromolecular complexes, such as Drosophila large ribonucleoprotein complexes and herpesvirus nucleocapsids, utilize a unique vesicle-mediated nucleocytoplasmic transport: the complexes acquire primary envelopes by budding through the inner nuclear membrane into the space between the inner and outer nuclear membranes (primary envelopment), and the enveloped complexes then fuse with the outer nuclear membrane to release de-enveloped complexes into the cytoplasm (de-envelopment). However, there is a lack of information on the molecular mechanism of de-envelopment fusion. We report here that HSV-1 recruited cellular fusion regulatory proteins CD98hc and β1 integrin to the nuclear membrane for viral de-envelopment fusion. This is the first report of cellular proteins required for efficient de-envelopment of macromolecular complexes during their nuclear egress.

FIG 1

Coimmunoprecipitation of CD98hc with HSV-1 gB in HSV-1-infected cells. (A and B) HEp-2 cells infected with wild-type HSV-1(F) at an MOI of 5 (3 × 10⁷ PFU/ml) for 24 h were harvested, immunoprecipitated (IP) with anti-Myc (α-Myc), anti-gB (α-gB), or anti-gC (α-gC) antibody, and analyzed by immunoblotting (IB) with anti-CD98hc (α-CD98hc) (A and B), anti-gB (A), or anti-gC (B) antibody. Molecular mass markers (in kilodaltons) are shown on the left. WCE, whole-cell extract.

FIG 2

Coimmunoprecipitation of CD98hc with HSV-1 gB, gH, UL31, UL34, and Us3, and β1 integrin in HSV-1-infected cells. (A) Schematic of the genome of wild-type HSV-1(F) (line 1) and of the MEF-CD98hc domain of recombinant virus YK715 (MEF-CD98hc) (line 2). (B) Characterization of YK715 (MEF-CD98hc). Vero cells were infected with wild-type HSV-1(F) or YK715 (MEF-CD98hc) at an MOI of 5 (5 × 10⁶ PFU/ml), and the total virus from cell culture supernatants and infected cells was harvested and assayed on Vero cells. (C) HEp-2 cells infected with wild-type HSV-1(F) or YK715 (MEF-CD98hc) at an MOI of 5 (3 × 10⁷ PFU/ml) for 24 h were harvested, immunoprecipitated with anti-Myc antibody, and analyzed by immunoblotting with the indicated antibodies.

FIG 3

Interaction of CD98hc with gB and gH. 293T cells were cotransfected with pcDNA-MEF-CD98hc and either pPEP-gB, pPEPgH, and pPEP-gL, or pPEP-gD. At 2 days posttransfection, the cells were harvested, immunoprecipitated with anti-Flag antibody, and analyzed by immunoblotting with the indicated antibodies. Molecular mass markers (in kilodaltons) are shown on the left. A longer exposure is also shown for gH.

FIG 4

Effect of HSV-1 infection on localization of CD98hc. HEp-2 cells were mock infected (A and B) or infected with wild-type HSV-1(F) (A to E) or YK538 (MEF-UL34) (F) at an MOI of 5 (5 × 10⁶ PFU/ml), fixed at 24 h postinfection, permeabilized, stained with the indicated antibodies, and examined by confocal microscopy. Bars, 10 μm.

FIG 5

Characterization of sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells. (A) The expression of CD98hc in sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells was analyzed by immunoblotting with anti-CD98hc (top) and anti-α-tubulin (bottom) antibodies. Molecular mass markers (in kilodaltons) are shown on the left. (B) The cell viability of sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells was assayed 24 h after 2 × 10⁴ cells were seeded on 96-well plates. Each value is the mean ± the standard error of the results of three independent triplicate experiments and is expressed relative to the mean for sh-Luc-HEp-2 cells, which was normalized to 100%. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey's test. n.s., not statistically significant.

FIG 6

Effect of CD98hc on localization of UL31 and UL34 in HSV-1-infected cells. (A) sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 5 (5 × 10⁶ PFU/ml), fixed at 24 h postinfection, permeabilized, stained with anti-UL34 and anti-UL31 antibodies, and examined by confocal microscopy. Bars, 10 μm. (B) sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 5 (5 × 10⁶ PFU/ml), fixed at 24 h postinfection, permeabilized, stained with anti-UL34 and anti-UL31 antibodies, and examined by confocal microscopy as described for panel A. The percentage of cells with aberrant punctate structures at the nuclear rim was determined. Each value is the mean ± the standard error of the results of three independent experiments. Statistical analysis was performed by one-way ANOVA with Turkey's test. We note that the standard errors are too small to be displayed.

FIG 7

Effect of CD98hc on HSV-1 nuclear egress. (A) sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 10 (10⁷ PFU/ml), fixed at 24 h postinfection, embedded, sectioned, stained, and examined by transmission electron microscopy. Arrowheads indicate invagination structures containing primary enveloped virions. Nu, nucleus; Cyt, cytoplasm; Nm, nuclear membrane. Bars, 500 nm. (B) sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 10 (10⁷ PFU/ml), fixed at 24 h postinfection, embedded, sectioned, stained, and examined by transmission electron microscopy as described for panel A. The percentage of enveloped perinuclear virions was determined.

FIG 8

Effect of HSV-1 infection on localization of β1 integrin. (A and B) HEp-2 cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5 (5 × 10⁶ PFU/ml), fixed at 24 h postinfection, permeabilized, stained with the indicated antibodies, and examined by confocal microscopy. Bars, 10 μm.

FIG 9

Characterization of sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells. (A) Expression of β1 integrin in sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells was analyzed by immunoblotting with anti-β1 integrin (top) and anti-α-tubulin (bottom) antibodies. Molecular mass markers (in kilodaltons) are shown on the left. (B) Cell viability of sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells assayed 24 h after 2 × 10⁴ cells were seeded on 96-well plates. Each value is the mean ± the standard error of the results of three independent triplicate experiments and is expressed relative to the mean for sh-Luc-HEp-2 cells, which was normalized to 100%. Statistical analysis was performed by one-way ANOVA and Tukey's test. n.s., not statistically significant.

FIG 10

Effect of β1 integrin on localization of UL31 and UL34 in HSV-1-infected cells. (A) sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 5 (5 × 10⁶ PFU/ml), fixed at 24 h postinfection, permeabilized, stained with anti-UL34 and anti-UL31 antibodies, and examined by confocal microscopy. Bars, 10 μm. (B) sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 5 (5 × 10⁶ PFU/ml), fixed at 24 h postinfection, permeabilized, stained with anti-UL34 and anti-UL31 antibodies, and examined by confocal microscopy as described for panel A. The percentage of cells with aberrant punctate structures at the nuclear rim was determined. Each value is the mean ± the standard error of the results of three independent experiments. Statistical analysis was performed by one-way ANOVA and Tukey's test.

FIG 11

Effect of β1 integrin on HSV-1 nuclear egress. (A) sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 10 (10⁷ PFU/ml), fixed at 24 h postinfection, embedded, sectioned, stained, and examined by transmission electron microscopy. Arrowheads indicate invagination structures containing primary enveloped virions. Nu, nucleus; Cyt, cytoplasm; NM, nuclear membrane. Bars, 500 nm. (B) sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 10 (10⁷ PFU/ml), fixed at 24 h postinfection, embedded, sectioned, stained, and examined by transmission electron microscopy as described for panel A. The percentage of enveloped perinuclear virions was determined.

FIG 12

Effect of CD98hc and on HSV-1 replication. (A to H) sh-Luc-HEp-2, sh-CD98hc-HEp-2, and sh-CD98hc/CD98hc-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 0.05 (5 × 10⁴ PFU/ml). At the indicated times postinfection, total virus from cell culture supernatants and infected cells (A, B, E, and F) and extracellular progeny virus from cell culture supernatants (C, D, G, and H) was harvested and assayed. Virus titers in panels A and C at 72 h postinfection and in panels E and G at 36 h postinfection are shown in panels B, D, F, and H, respectively. Each value is the mean ± the standard error of the results of three independent experiments. Statistical analysis was performed by one-way ANOVA and Tukey's test.

FIG 13

Effect of CD98hc and β1 integrin on influenza virus replication. (A) sh-Luc-HEp-2 and sh-CD98hc-HEp-2 cells were infected with influenza virus at an MOI of 0.01 (5 × 10⁴ PFU/ml). At the indicated times postinfection, extracellular virus from cell culture supernatants was harvested and assayed. (B) sh-Luc-HEp-2 and sh-β1 integrin-HEp-2 cells were infected with influenza virus at an MOI of 0.01. At the indicated times postinfection, extracellular virus from cell culture supernatants was harvested and assayed.

FIG 14

Effect of CD98hc and β1 integrin on HSV-1 replication. (A) sh-Luc-HEp-2, sh-β1 integrin-HEp-2, and sh-β1 integrin/β1 integrin-HEp-2 cells were infected with wild-type HSV-1(F) at an MOI of 0.05 (5 × 10⁴ PFU/ml). At the indicated times postinfection, extracellular virus from cell culture supernatants were harvested and assayed. Each value is the mean ± the standard error of the results of three independent experiments. (B) Virus titers in panel A at 72 h postinfection. Each value is the mean ± the standard error of the results of three independent experiments. Statistical analysis was performed by one-way ANOVA and Tukey's test.

FIG 15

Effect of HSV-1 infection on αV and α5 integrins. (A) HEp-2 cells infected with wild-type HSV-1(F) or YK715 (MEF-CD98hc) at an MOI of 5 (3 × 10⁷ PFU/ml) for 24 h were harvested, immunoprecipitated with anti-Myc antibody, and analyzed by immunoblotting with the indicated antibodies. (B and C) HEp-2 cells were mock infected or infected with wild-type HSV-1(F) at an MOI of 5 (5 × 10⁶ PFU/ml), fixed at 24 h postinfection, permeabilized, stained with the indicated antibodies, and examined by confocal microscopy. Bars, 10 μm.