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. 2020 Jul 30;94(16):e00167-20.
doi: 10.1128/JVI.00167-20. Print 2020 Jul 30.

Human Cytomegalovirus Glycoprotein-Initiated Signaling Mediates the Aberrant Activation of Akt

Jamil Mahmud  1 , Michael J Miller  1 , Aaron M Altman  1 , Gary C Chan  2
Affiliations

Human Cytomegalovirus Glycoprotein-Initiated Signaling Mediates the Aberrant Activation of Akt

Jamil Mahmud et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality among immunocompromised and immunonaive individuals. HCMV-induced signaling initiated during viral entry stimulates a rapid noncanonical activation of Akt to drive the differentiation of short-lived monocytes into long-lived macrophages, which is essential for viral dissemination and persistence. We found that HCMV glycoproteins gB and gH directly bind and activate cellular epidermal growth factor receptor (EGFR) and integrin β1, respectively, to reshape canonical Akt signaling within monocytes. The remodeling of the Akt signaling network was due to the recruitment of nontraditional Akt activators to either the gB- or gH-generated receptor signaling complexes. Phosphoinositide 3-kinase (PI3K) comprised of the p110β catalytic subunit was recruited to the gB/EGFR complex despite p110δ being the primary PI3K isoform found within monocytes. Concomitantly, SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) was recruited to the gH/integrin β1 complex, which is critical to aberrant Akt activation, as SHIP1 diverts PI3K signaling toward a noncanonical pathway. Although integrin β1 was required for SHIP1 recruitment, gB-activated EGFR mediated SHIP1 activation, underscoring the importance of the interplay between gB- and gH-mediated signaling to the unique activation of Akt during HCMV infection. Indeed, SHIP1 activation mediated the increased expression of Mcl-1 and HSP27, two Akt-dependent antiapoptotic proteins specifically upregulated during HCMV infection but not during growth factor treatment. Overall, our data indicate that HCMV glycoproteins gB and gH work in concert to initiate an HCMV-specific signalosome responsible for the atypical activation of Akt required for infected monocyte survival and ultimately viral persistence.IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic throughout the world regardless of socioeconomic conditions and geographic locations with a seroprevalence reaching up to 100% in some developing countries. Although asymptomatic in healthy individuals, HCMV can cause severe multiorgan disease in immunocompromised or immunonaive patients. HCMV disease is a direct consequence of monocyte-mediated systematic spread of the virus following infection. Because monocytes are short-lived cells, HCMV must subvert the natural short life-span of these blood cells by inducing a distinct activation of Akt, a serine/theonine protein kinase. In this work, we demonstrate that HCMV glycoproteins gB and gH work in tandem to reroute classical host cellular receptor signaling to aberrantly activate Akt and drive survival of infected monocytes. Deciphering how HCMV modulates the cellular pathway to induce monocyte survival is important to develop a new class of anti-HCMV drugs that could target and prevent spread of the virus by eliminating infected monocytes.

Keywords: cytomegalovirus; monocytes.

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Figures

FIG 1
FIG 1
HCMV gB and gH stimulate Akt activity to promote the survival of HCMV-infected monocytes. (A) Peripheral blood monocytes were mock or HCMV infected or infected with the virus neutralized with antibody against glycoprotein gB or gH or isotype antibody for 15 min. The phospho (p)-Akt (Ser473) and total-Akt were detected by immunoblotting from whole-cell lysates. Actin was used as a loading control. Results are representative of 3 to 5 independent experiments using different donors. Densitometry analysis was performed using Image Lab software (Bio-Rad) and quantification of the specific blots shown is marked as “donor 1,” and data points from another donor (donor 2) have additionally been provided to account for donor variability. (B, C, and D) Monocytes were infected with mock or nonneutralized HCMV or HCMV neutralized with antibody against glycoprotein gB or gH or isotype antibody and incubated for 48 h. Viability was measured by annexin V and propidium iodide (PI) staining using flow cytometry. (B) Monocytes negative for both annexin and PI were considered live cells. (C and D) Lines connect data points from the same experiment using the same donor. Results are representative of 3 to 5 independent experiments using different donors. Statistical significance was measured using paired t test; *, P < 0.05; **, P < 0.005; ns, nonsignificant.
FIG 2
FIG 2
Soluble sgB and sgH induce monocyte survival via activation of Akt. (A) Schematic representation of the full-length HCMV glycoprotein gB or gH along with the soluble sgB and sgH construct. SS, signal sequence; TMD, transmembrane domain; CPD, cytoplasmic domain; His, histidine tag. (B) HCMV virus particles were lysed and immunoblotted for gB and gH. Soluble His-tagged sgB and sgH were purified from the supernatants of stably transduced Expi293F cells by nickel column purification and immunoblotted for gB, gH, or His tag. (C, D, and E) Monocytes were mock or HCMV infected or treated with sgB (1 μg/ml) and/or sgH (1 μg/ml) or Expi293 cell supernatant negative control (NC). (C) After 15 min, p-Akt (Ser473) and total Akt levels were measured by immunoblotting. (D and E) Monocyte viability was measured after 48 h by annexin V and PI staining using flow cytometry. Results are representative of 3 to 5 independent experiments using different donors. *, P < 0.05; ns, nonsignificant.
FIG 3
FIG 3
Glycoproteins gB and gH directly bind to and activate EGFR and integrin β1, respectively. (A, B, and C) Monocytes were mock or HCMV infected or treated with sgB (5 μg/ml) or sgH (5 μg/ml) for 0 min or 5 min (A) or for 0 min (B and C) and immunoprecipitated with antibodies recognizing gB, gH, EGFR, integrin β1, or isotype antibody. Western blot analyses were performed to determine the presence of gB, gH, EGFR, p-EGFR, integrin β1, p-integrin β1, and integrin β3 in the immunoprecipitated samples. Input controls were blotted for actin to confirm homogeneous loading of the samples. (B) Immunoblot is from a single gel from the same experiment. (A, B, and C) Results are representative of at least 3 independent experiments using different donors.
FIG 4
FIG 4
Glycoproteins gB and gH activate Akt through EGFR and integrin β1 during monocyte entry. (A) Monocytes were mock or HCMV infected or treated with sgB or sgH. After 15 min, p-EGFR and p-integrin β1 levels were measured by immunoblotting. (B, C, D, E, and F) Monocytes were mock or HCMV infected or treated with sgB, sgH, or negative control (NC). Indicated samples were preincubated with 5 μM AG (an EGFR inhibitor) and 20 μM ATN (an integrin inhibitor) for 1 h at 37°C prior to infection or treatment. (B, C, and D) After 15 min, cells were lysed and levels of p-EGFR, p-integrin β1, p-Akt, and total Akt were measured by immunoblotting. (E) After 48 h, monocyte viability was measured by annexin V and PI staining using flow cytometry. (F) Mcl-1 and HSP27 were detected after 48 h by immunoblotting from whole-cell lysates. Results are representative of at least 3 independent experiments using different donors. *, P < 0.05; **, P < 0.005.
FIG 5
FIG 5
Phosphorylation of EGFR and integrin β1 results in the activation of the EGFR/PI3K/SHIP1 signaling pathway. (A and B) EGFR, integrin β1, p85, and SHIP1 were immunoprecipitated after monocytes were infected with or without HCMV for 0 min or 5 min (A) or 5 min (B). (C) Cells were treated with 5 μM AG (an EGFR inhibitor) and 20 μM ATN (an integrin inhibitor) for 1 h at 37°C and then infected with HCMV for 15 min. Cells were then lysed followed by Western blot analyses to determine the presence of different proteins using specific antibodies. Results are representative of at least 3 independent experiments using different donors.
FIG 6
FIG 6
HCMV induces a chronic SHIP1 activation required for the survival of infected monocytes. (A) Monocytes were mock or HCMV infected for 15 min, 24 h, or 48 h, and total or p-SHIP1 was detected from whole-cell lysates. (B, C, and D) Monocytes were treated with 5 μM AG (an EGFR inhibitor), 50 μM LY (a PI3K inhibitor), or 5 μM 3AC (a SHIP1 inhibitor) for 1 h and then mock or HCMV infected. (B) After 5 min infection, anti-gH antibody was used to pull down gH glycoprotein (and any other proteins bound to gH) followed by blotting for gH, integrin β1, SHIP1, and p-SHIP1 in the immunoprecipitate using specific antibodies. Input controls were blotted for actin to confirm homogeneous loading of the samples. (C) After 24 h, Western blot analysis was performed to detect Mcl1 and HSP27 from total cell lysates. (D) Survival of monocytes was measured after 48 h of infection with or without the inhibitors by annexin V and PI staining using flow cytometry. Results are representative of at least 3 to 5 independent experiments using different donors. **, P < 0.005.
FIG 7
FIG 7
Proposed model for activation of monocyte receptors by HCMV glycoproteins required for Akt phosphorylation and subsequent survival of infected monocytes. During HCMV entry into monocytes, gB and gH directly bind EGFR and integrin β1, respectively. Activation of either receptor can also lead to the cross-activation of the other receptor. Ultimately, activation of EGFR by HCMV results in the recruitment of the p85 and p110β subunits of PI3K to initiate signaling from PI(3,4,5)P3. However, the concomitant recruitment of SHIP1 to integrin β1 diverts signaling away from canonical PI(3,4,5)P3 signaling to a noncanonical PI(3,4)P2-mediated activation of Akt. The nonclassical activation of Akt leads to the upregulation of a select subset of Akt-dependent antiapoptotic proteins required for the survival of HCMV-infected monocytes.
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