Hek293t cells

Lentiviral plasmid was packaged as previously described (Xie et al., 2019 (link)). Briefly, the lentivirus packaging plasmids PMD2.G and psPAX2 (Addgene ID: 12259 and 12260) were co-transfected with the carrier plasmid to HEK293T cells with linear polyethylenimine (Polysciences). Supernatant was collected 72 hr after transfection and filtered with a 0.45 μm filter. The virus was further purified by using Lenti-X lentivirus concentrator (Clontech).

3 × 10⁶ HEK293T cells (ATCC) were seeded per 10‐cm dish and transfected with 12.5 μg of DNA the following day (4.95 μg of pL expression constructs, 4.95 μg pCMV‐dR8.92 (a gift from Bob Weinberg via Addgene (plasmid #8455)) (Stewart et al, 2003), 1.45 μg pMD2.G, and 1.15 μg pRSV‐Rev (both from Didier Trono via Addgene (plasmid #12253 and #12259) (Dull et al, 1998)) using polyethyleneimine (PEI, 1 mg/ml; PolySciences) at a ratio of 1:3 (DNA:PEI). Cells were incubated for 72 h, and virus particles were harvested by ultracentrifugation of the cell culture supernatants (90 min at 65,000 g, rotor SW‐28, Optima^™ L‐100 K Ultracentrifuge, Beckman Coulter). Viral pellets were resuspended in 1% BSA/PBS, and aliquots were stored at −80°C. In order to titer the viruses, HeLa cells were seeded into 12‐well plates at a density of 75,000 cells per well and transduced with 500 μl of a 10⁻², 10⁻³, and 10⁻⁴ dilution of the virus the next day. After 72 h, cells were trypsinized and fixed (4% formaldehyde/PBS) and EGFP or mVenus fluorescence was analyzed by fluorescence‐activated cell sorting (FACS; BD^™ LSR II Flow Cytometer). The following formula was used to calculate the number of TU/ml: number of cells before transduction/percentage of positive gated cells × dilution factor × 2.

We used the following previously published cell lines: HEK293T cells (American Type Culture Collection, cat. no. CRL-3216), hTERT immortalized RPE‐1 (RPE1) wild-type cells (American Type Culture Collection, cat. no. CRL-4000), RPE1 AKAP450 knockout and RPE1 AKAP450/CDK5RAP2/MMG triple knockout cell lines^{47 (link)}, and RPE1 wild-type and RPE1 AKAP450 knockout transgenic cell lines stably expressing GFP–CDK5RAP2 (ref. ^{72 (link)}). All these cell lines were cultured in Dulbecco’s modified Eagle medium/Ham’s F10 media (1:1) supplemented with 10% fetal calf serum and 1% antibiotics (penicillin and streptomycin). The cell lines used were not found in the commonly misidentified cell lines database maintained by the Immunization Coalition of Los Angeles County. No further cell line authentication was performed. The cell lines were routinely checked for mycoplasma contamination using the LT07–518 Mycoalert assay. Polyethylenimine ‘Max’ (PEI Max, Polysciences) was used to transfect HEK293T cells with plasmids for Strep-Tactin- and streptavidin-based protein purification at a 3:1 ratio of PEI Max:plasmid.

The pCDNA-GFP-Rab5 clone was provided by Dr. Yoshikatsu Aikawa at Doshisha University, Japan. The pGEX-EEA1_36-91 clone was obtained from Dr. Bonsu Ku at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). cDNA clones of Rabaptin5 and Rabex-5 were purchased from Open Biosystems. The Rab7 gene was chemically synthesized (Bioneer). Each gene was amplified and cloned into bacterial expression vectors (parallel-His, parallel-GST (Sheffield et al., 1999 (link)), and pQE30) or mammalian expression vectors (pCDNA-FLAG and pCDNA-HA). Human embryonic kidney 293T (HEK 293T) and HeLa cells were purchased from American Type Culture Collection (RRID: CVCL_0063 and CVCL_0030, respectively) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (heat-inactivated). Plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) for HeLa cells and PEI 25000 (23966, Polysciences) for HEK 293T cells. Mycoplasma detection kit (LT07-318, Lonza, Switzerland) was regularly used to check absence of mycoplasma.

A nonmuscle-type myosin light chain (MRLC12A) cDNA was obtained from a human kidney cDNA library and transferred to pEB Multi vector (Fujifilm Wako Chemicals). HEK293T cells (Invitrogen) were transfected with the vector DNA, and neomycin-resistant MRLC12A-expressing HEK293T cells were created. EGFP-HM19^FullLZ was then transiently expressed in MRLC12A-expressing HEK293T cells by a transfection of recombinant Myo19 heavy chain cDNAs using PEI Max (Polysciences, Inc) and then purified using anti-FLAG antibody-agarose resins (SIGMA). In short, 6 × 10⁷ cells were harvested at 24 h after transfection and homogenized in a lysis buffer (0.25 M KCl, 50 mM Hepes–KOH [pH 7.5], 5 mM EGTA, 15 mM β-mercaptoethanol, 1 mM ATP, 2% CHAPS, and protease inhibitor cocktail [described previously]). The suspension was mixed with anti-FLAG antibody-agarose resins, and the tube containing the suspension was rotated at 4 °C for 1 h. After two washes with a wash buffer (0.12 M KCl, 12.5% trehalose, 1 mM EGTA, 15 mM β-mercaptoethanol, 2% CHAPS, and protease inhibitor cocktail), the protein was eluted with an elution buffer (the wash buffer containing 0.1 mg/ml FLAG peptide). The eluted fractions were flash-frozen by liquid nitrogen and stored at −80 °C.

SARS-CoV-2 pseudoviruses were produced as previously described^{78 (link),79 (link)}. Briefly, the backbone plasmid pNL4-3.Luc.R-E and pCAGGS-S-CΔ19 expressing spike of the WT strain or its variants were cotransfected into HEK 293 T cells by polyethyleneimine (Polysciences, Warrington, PA, USA). The spike amino acid sequences of WT strain and its variants were based on GISAID EPI_ISL_402124 (Wuhan/WIV04/2019), EPI_ISL_712096 (Beta variant), EPI_ISL_2029113 (Delta variant), EPI_ISL_6640916 (BA.1 variant), EPI_ISL_8515362 (BA.2 variant), EPI_ISL_13241867 (BA.5 variant), EPI_ISL_15812431 (BQ.1 variant), or EPI_ISL_14917761 (XBB.1 variant). The supernatant was collected 48 h post-transfection, filtered through 0.45 µm filters, and concentrated overnight with PEG 8000 at 4 °C.
Pseudovirus titration was performed based on HIV-1 p24 antigen quantification utilizing Lentivirus Quantitation Kit (Beijing Biodragon Immunotechnologies Co., Ltd, Beijing, China) following the manufacturer’s instructions; 1 × 10⁸ lentivirus particles per well were used for the pseudovirus neutralization test.