Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release

Abstract

Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4B^C74A). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4B^C74A, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment.Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short.

Keywords: Cellular host defense; herpesviruses; human cytomegalovirus; viral morphogenesis; xenophagy.

Figure 1.

LC3 and SQSTM1 localized to viral assembly compartments. Confocal immunofluorescence analysis of LC3 and SQSTM1, respectively (Alexa Fluor 488, green), and of the viral protein pp150 (Alexa Fluor 546, red). Cells were infected for 6 d with the laboratory strain BADwt at an m.o.i. = 0.2. Uninfected cells (mock) were carried along for control. Cells were then pre-incubated with goat antiserum to block unspecific binding of the antibodies. Rabbit monoclonal anti-LC3 or anti-SQSTM1 antibodies were used for staining. The mouse monoclonal pp150 antibody was used for staining of the cVACs. Both LC3 and SQSTM1 localize to cytoplasmic virus assembly compartments and colocalize with pp150

Figure 2.

SQSTM1 localized to intranuclear viral capsids. Immunoelectron micrographs of late-stage-infected fibroblasts. Fixed samples of HFF cells, infected with BADwt at an m.o.i. = 0.8 for 6 d were incubated with a 1:1 mix of two mouse monoclonal antibodies directed against MCP and sCP (A, B), respectively, or with a rabbit monoclonal antibody directed against anti-SQSTM1 (C-F), respectively. A secondary antibody, conjugated with gold particles and silver-enhanced, was used. Nucleus (N) and cytoplasm (C) are indicated. (A) Gold particles accumulate in the nucleus, labeling the viral capsids present in the nucleus of an HCMV–infected fibroblast (indicated by arrows). (B) is an enlarged version of the indicated field from (A). (C-F) SQSTM1 localizes to intranuclear viral capsids (indicated by arrows). (D, E, and F) display enlarged sections of (C)

Figure 3.

LC3 and SQSTM1 localized to cytoplasmic viral particles. Immunoelectron micrographs of late-stage-infected fibroblasts. HFF cells, infected with BADwt at an m.o.i. = 0.8 for 6 d were co-stained with a 1:1 mix of anti-MCP and anti-sCP mouse monoclonal antibodies and either an anti-SQSTM1 antibody (A, B) or an anti-LC3 monoclonal antibody (C, D), both derived from rabbit, respectively. A secondary antibody, conjugated with gold particles and silver-enhanced, was used. Secondary antibodies against mouse immunoglobulin (MCP/sCP) were conjugated to smaller gold particles, antibodies against rabbit immunoglobulin (LC3, SQSTM1) to larger gold particles. Arrows indicate HCMV virions in the cytoplasm of an infected fibroblast. Both SQSTM1 (A, B) and LC3 (C, D) colocalize with MCP/sCP at viral particles in the cytoplasm. Mitochondria (M) and subviral Dense Bodies (DB) are indicated

Figure 4.

Lipidated LC3 is present in purified viral particles. Western blot analysis of whole-cell lysates of uninfected HFF and 30 µg of purified virions, respectively, and probed with antibodies against MCP, SQSTM1, and LC3B. Three different laboratory strains were tested. Only the lipidated form of LC3, LC3-II, was found in HCMV virions

Figure 5.

SQSTM1 co-purified with the viral capsid and components of the viral tegument. Immunoprecipitation of BADwt-infected HFF with mouse anti-SQSTM1 antibody and empty magnetic beads as control, and protein quantification by mass spectrometry. We identified 41 HCMV protein groups, 28 of which could be quantified in both IPs and technical replicates. (A) Log₂ transferred average fold-change of SQSTM1, and viral component proteins are plotted with the standard error of the mean (SE), indicated as error bars. Values are calculated for two independent IPs (biological replicates) with two technical replicates in each case. (B) A Welch’s t-test was performed between the mean of all viral proteins and all human background proteins, with a fold-change between 0.5 and 2. The bar chart represents the average of all viral proteins and human background proteins. Error bars indicating the standard deviation (SD). (C) Immunoprecipitation of BADwt-infected HFF with mouse anti-SQSTM1 antibody and mouse IgG as control. Eluates and supernatants were subjected to SDS protein-gel separation, and western blot probed with antibodies against SQSTM1, pp28, and sCP/pUL48a. sCP/pUL48a specifically precipitates with SQSTM1, while pp28 does not. Unbound sCP/pUL48a and pp28 were still present in the cell lysates after immunoprecipitation. SQSTM1 was no longer detectable by western blot in the supernatant of the specific SQSTM1 precipitate. In the control supernatant, the amounts of SQSTM1 were comparable to input levels

Figure 6.

SQSTM1 colocalized with the major capsid protein. Confocal immunofluorescence analysis of late-stage-infected HFF. Cells were infected with laboratory strain BADwt (A) or with strain TB40/E (B) at an m.o.i. of 0.2. After 6 d, cells were then pre-incubated with goat antiserum to block unspecific binding of the antibodies. Staining was performed with antibodies against SQSTM1 (Alexa Fluor 488, green) and antibodies against the viral proteins MCP or pp28, respectively (Alexa Fluor 546, red). SQSTM1 colocalizes with viral MCP (yellow), but not with the tegument protein pp28 (control)

Figure 7.

Infection with HCMV strain BAD-ATG4B^C74A eliminated the lipidation of LC3. (A) Prior to lipidation, a precursor form of LC3, pro-LC3, is cleaved by the protease ATG4B. This step is essential for the subsequent membrane conjugation event of LC3. A dominant-negative version of ATG4B does not cleave pro-LC3, and also inhibits the interaction of LC3-I with ATG7, thereby preventing the lipidation of LC3-I. (B) Schematic presentation of an HCMV strain, expressing a dominant-negative version of ATG4B, termed BAD-ATG4B^C74A. The generation of this virus was performed using the galK recombination method, producing a 2 USD-11-deleted intermediate BACmid. In this BACmid, the gene region 2 USD-11 of the parental strain BADwt was replaced by a DNA fragment, encoding the bacterial galK gene, required for positive selection in bacteria. In a second step, a DNA fragment encoding a hemagglutinin antibody epitope (HA)-tagged version of ATG4B^C74A (HA-ATG4B^C74A) was used to replace the galK gene. Both this BAC and the intermediate BAC-∆2 USD-11 were reconstituted, resulting in the viruses BAD-∆2 USD-11 (control) and BAD-ATG4B^C74A. In the latter virus, the ATG4B^C74A construct is driven by a modified version of the major immediate-early promoter of HCMV, containing a nonfunctional cis repressive sequence (crs). (C) Western blot analysis of HFF infected with BAD-ATG4B^C74A for different times. The expression of ATG4B^C74A starts at day 1 p.i. and also continues at later stages of infection. (D) Western blot analysis of LC3 expression in HFF infected with BADwt, BAD-∆2 USD-11, or BAD-ATG4B^C74A, respectively, for different times. Mock-infected cells were taken along for reference. While BADwt and BAD-∆2 USD-11 induce the lipidation of LC3 (LC3-II) to a similar extent at 24 h.p.i., LC3-II is reduced and the amounts of non-lipidated LC3-I increase on day 2 post-infection onwards with BAD-ATG4B^C74A. (E) Western blot analysis of purified viral particles of BADwt and BAD-ATG4B^C74A. Virions of BAD-ATG4B^C74A contain neither form of LC3, as opposed to purified virions of the parental strain BADwt, which display the membrane-bound form LC3-II. Both viruses contain similar amounts of SQSTM1

Figure 8.

Cytopathic effect and progeny release are enhanced in BAD-ATG4B^C74A-infected HFF. (A) Transmission microscopy of HFF infected with 0.25 genomes/cell of either BADwt, BAD-∆2 USD-11, or BAD-ATG4B^C74A, respectively. The CPE of infected cells was documented at various time points. (B) Kinetics of virus release from HFF used in (A), infected with 0.25 genomes/cell of either BADwt, BAD-∆2 USD-11, or BAD-ATG4B^C74A. Culture supernatants were collected at the indicated time points and analyzed by 8 technical replicates, using serial dilution and counting numbers of indicator cells, stained for the immediate-early 1 protein (IE1) of HCMV

Figure 9.

Viral DNA replication and release of progeny genomes are enhanced in BAD-ATG4B^C74A-infected HFF. (A) Quantitative PCR analysis of the levels of viral genomes present in infected cells at different times after infection. HFF cells were infected with BADwt, BAD-∆2 USD-11, or BAD-ATG4B^C74A with 0.25 genome copies/cell, respectively. Infection conditions were chosen in a way to ensure that equal genome copy numbers were initially present in the cells (at 4 h.p.i.). The values of the three technical replicates are shown. Cells were collected at the indicated time points, lysed, and subjected to PCR analysis. Copy numbers are indicated at selected time points. (B) Quantitative PCR analysis of viral genomes present in the cell media of the cultures used in (A). Samples were taken at the indicated time points and subjected to PCR analysis. Copy numbers are indicated at selected time points

Figure 10.

Hypothetical model of autophagy and viral morphogenesis as simultaneous, competing processes. HCMV capsids are assembled in the cell nucleus. The inner tegument protein pp150 covers the viral capsid in the nucleus to stabilize it. SQSTM1 is present in the nucleus and interacts with capsids (A). Capsids are transported through the nuclear membrane by the process of envelopment and de-envelopment (B). Capsids are transported to cVACs, where they are decorated with proteins of the outer tegument (C). One or more of these tegument proteins may interact with SQSTM1 or other autophagy receptor proteins, possibly via ubiquitin or other small modifiers, enabling interaction with LC3-II-conjugated membranes to foster envelopment (D). How membranes of other sources merge into this process is unclear at this point. Vesicles containing one or more viral particles are then transported to the cell periphery (E), where they fuse with the plasma membrane to release viral particles (F). The findings that capsid proteins precipitate with SQSTM1 and that SQSTM1 interacts with viral capsids already in the nucleus, foster the hypothesis that capsids and attached inner tegument proteins are tagged with autophagy receptor proteins (G) to label them for autolysosomal degradation (H, I). The inhibition of autophagy may lead to an increase in “tegumented” capsids, and other membrane sources may compensate for the loss of LC3-positive membranes in the process of HCMV secondary envelopment