Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection

Abstract

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we showed that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required during flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrated specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical and imaging methodology, we showed that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT proteins, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection, the virus employs members of ESCRT proteins for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Another important ESCRT protein, TSG101, is not required for virus production. The ESCRT, complex, ALIX-CHMP4A, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

Keywords: ALIX; CHMP4A; ESCRT; HERC5; ISG15; NS3; TSG101; assembly; envelope; replication; replicons; tick-borne flaviviruses; virus late domain.

FIG 1

ESCRT proteins ALIX and CHMP4A are essential for virus production. (A) Immunoblotting of A549 cell lysates was performed 24–48 h posttransfection with the antibody against either CHMP4A, TSG101, or ALIX after specific siRNA treatments. The expression levels were normalized using the endogenous proteins, either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin. (B) Cytotoxicity assay was performed by measuring the amount of lactate dehydrogenase (LDH) enzyme released into cell culture media during siRNA treatments. When the plasma membrane is damaged, the endogenous LDH is released into media and reacts to substrates from the assay, generating a red formazan product that can be measured spectrophotometrically at 490 nm and normalized to the absorbance at 680 nm. (C) The virus titers in supernatants from the TSG101, ALIX, or CHMP4A siRNA-treated cells were compared to the NT siRNA-treated cells after Langat virus (LGTV), West Nile virus strain Kunjin (WNV_Kunjin), or Zika virus (ZIKV), Powassan virus (POWV), Kyasanur forest disease virus (KFDV), and tick-borne encephalitis virus strain Sofjin (TBEV_Sofjin) infection. The virus titers were measured by plaque assay. (D) The virus genome copy numbers were measured by qPCR from supernatants of the indicated siRNA-treated cells. The experiments were conducted independently three times with two technical repeats. The P values are indicated using * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

FIG 2

Reduction of intracellular virus particles and virus genome copy numbers during ALIX or CHMP4A depletion. (A) Virus titers in cell lysates from TSG101, ALIX, or CHMP4A siRNA-treated cells were compared to the control NT siRNA-treated cells after LGTV, WNV_Kunjin, ZIKV infection. The virus titers were measured by plaque assay. (B) Virus genome copy numbers were measured by qPCR from cell lysates of indicated siRNA-treated cells. The experiments were conducted independently three times with two technical repeats. The P values are indicated using * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

FIG 3

Roles of the ESCRT proteins during flavivirus infection. (A) Schematic illustration of the T7 driven RNA replicon construct comprising: 5′-untranslated region (UTR), the firefly luciferase gene (Luc) as a reporter gene substituted for most genes coding the structural proteins, the foot-and-mouth disease virus autoprotease 2a (FMDV 2A), all the nonstructural proteins, the 3′-UTR, the antigenomic hepatitis delta virus ribozyme (HDVr) sequence, and the simian virus 40 (SV40) polyadenylation signal (pA). An internal ribosome entry site (IRES) sequence and the neomycin/kanamycin resistance (NeoR/KanR) gene were inserted inside the 3′-UTR. (B) Immunoblotting of baby kidney hamster 21 (BHK-21) cell lysates 24–48 h posttransfection with antibodies against either CHMP4A, TSG101, or ALIX after specific siRNA treatments. The expression levels were normalized using the endogenous protein GAPDH. (C) Luminescent units from the BHK21 cell lysates expressing the WNV_Kunjin or the LGTV replicons during TSG101, ALIX, or CHMP4A silencing in comparison with the control. (D) Replicon genome copy numbers measured by qPCR from the indicated siRNA-treated cells. (E) Luminescent units from the naive BHK-21 cells infected by the replicon virus-like particles (RVPs) during indicated siRNA treatments compared to the control. The experiments were conducted independently three times with two technical repeats. The P values are indicated using * P < 0.05, ** P < 0.01, and **** P < 0.0001.

FIG 4

ALIX is recruited to the virus replication sites. Immunostaining of A549 cells 48h after infection with LGTV. (A) The cells were labeled with anti dsRNA (red) and ALIX (green), while in (B) the cells were labeled with anti E (red) and ALIX (green). The nuclei were counterstained with DAPI (blue). The insets represent images from the yellow squares. The arrowhead points to the area where there is high colocalization of red and green fluorophores, resulting in yellow fluorophores. Bar scales represent 20 μm. (C) and (D) Graphs illustrate green and red fluorescent intensity at the arrow in the panel (A) and (B), respectively. Stars indicate the overlapping peaks of fluorescent intensity.

FIG 5

Close localization and interaction between the ESCRT proteins and the TBEV_Toro NS3 proteins. (A) Immunostaining of A549 cells transfected with the HA-NS3 or HA-PAR6 construct. The cells were labeled with anti HA (red) and ALIX (green). The nuclei were counterstained with DAPI (blue). The insets represent images from the yellow squares. The arrowhead points to the area where there is high colocalization of red and green fluorophores, resulting in yellow fluorophores. Bar scales represent 20 μm. (B) Graphs illustrate green and red fluorescent intensity at the arrow in the panel (A). Stars indicate the overlapping peaks of the fluorescent intensity. (C) Schema illustrates the conserved late domain motif LYXXA inside the NS3 protein beginning at the 1795^th amino acid from the first methionine of the polyprotein, using TBEV_Toro as a reference virus. (D) Schema illustrates another putative L domain sequence YPTI within the YFV NS3 protein as described in (24). (E) Immunoblotting of cell lysates 48 h posttransfection with HA-PAR6, HA-NS5, HA-NS3, or HA-NS3 Y1796L T1797L constructs, and the immunoprecipitation (IP) eluates by an anti-HA antibody. The proteins were visualized with the antibodies against HA, ALIX, CHMP4A, and GAPDH as the loading control. (F) Immunoblotting of cell lysates 48 h posttransfection with mCherry, prM-E-mCherry constructs, and the eluates after pulling down by an anti-mCherry antibody. The proteins were visualized with the antibodies against mCherry, ALIX, CHMP4A, and GAPDH as the loading control. (G) Relative luminescent units (RLUs) from the A549 cells transfected with a renilla luciferase-expressing construct and either DNA TBEV_Toro replicon, TBEV_Toro replicon with Y1796L T1797L mutations, or truncated TBEV_Toro replicon with a stop codon inserted in NS5 (35).

FIG 6

ISG15 expression enhanced effects of ALIX or CHMP4A depletion. (A) LGTV titers from supernatants of A549 cells transfected with either ISG15 or GFP constructs and with indicated siRNA, measured by plaque assay. (B) LGTV genome copy numbers from lysates of A549 transfected with indicated constructs and siRNA, measured by qPCR. (C) Expression of luciferase reporter from the BHK-21 cell line expressing the LGTV replicon and transfected with either GFP, ISG15 or HERC5 constructs. The experiments were conducted independently three times with two technical repeats. The P values are indicated using * P < 0.05, ** P < 0.01, and *** P < 0.001.

FIG 7

Expression of ISG15 and HERC5 resulted in degradation of TSG101, ALIX, and CHMP4A. (A) Immunoblotting of supernatants and cell lysates transfected with either GFP, ISG15, or HERC5 constructs and treated with either NT or TSG101 siRNA. The cells were then infected with LGTV. The proteins were visualized with the antibodies against GFP, ISG15, HERC5, TSG101, ALIX, CHMP4A, the 94 kDa glucose-regulated protein (GRP94), E, prM, and GAPDH as the loading control. (B), (C), (D), and (E) Relative intensity of the ALIX, TSG101, or GRP94 protein bands from the blots in (A). The experiments were conducted independently three times. The P values are indicated using *** P < 0.001.

FIG 8

Schema illustrates interplays between the ESCRT proteins (ALIX, CHMP4A) and the ISG15/HERC5. ALIX has roles in virus replication and CHMP4A is required for virus assembly. To counteract virus infection, infected cells induce the ISG15/HERC5 system to degrade the ESCRT proteins. The schema was created using Biorender web tool.