viruses

Article Identification of the SHREK Family of as Broad-Spectrum Host Antiviral Factors

Deemah Dabbagh, Sijia He , Brian Hetrick, Linda Chilin, Ali Andalibi and Yuntao Wu *

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, VA 20110, USA; [email protected] (D.D.); [email protected] (S.H.); [email protected] (B.H.); [email protected] (L.C.); [email protected] (A.A.) * Correspondence: [email protected]

Abstract: and -like molecules are highly glycosylated, high-molecular-weight cell surface proteins that possess a semi-rigid and highly extended extracellular domain. P- ligand-1 (PSGL-1), a mucin-like glycoprotein, has recently been found to restrict HIV-1 infectivity through virion incorporation that sterically hinders virus particle attachment to target cells. Here, we report the identification of a family of antiviral cellular proteins, named the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators (PSGL-1, CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123) that share similar structural characteristics with PSGL-1. We demonstrate that SHREK proteins block HIV-1 infectivity by inhibiting virus particle attachment to target cells. In addition, we demonstrate that SHREK proteins are broad-spectrum host antiviral factors that block the infection of diverse viruses such as influenza A. Furthermore, we demonstrate that a subset of SHREKs also blocks the infectivity of a hybrid alphavirus-SARS-CoV-2  (Ha-CoV-2) pseudovirus. These results suggest that SHREK proteins may be a part of host innate  immunity against enveloped viruses. Citation: Dabbagh, D.; He, S.; Hetrick, B.; Chilin, L.; Andalibi, A.; Keywords: SHREK; PSGL-1; CD34; PODXL1; PODXL2; CD164; MUC4; TMEM123; HIV-1; Influenza Wu, Y. Identification of the SHREK A; SARS-CoV-2; Ha-CoV-2 Family of Proteins as Broad-Spectrum Host Antiviral Factors. Viruses 2021, 13, 832. https://doi.org/ 10.3390/v13050832 1. Introduction Mucins and mucin-like molecules are highly glycosylated, high-molecular-weight cell Academic Editor: Mariana Baz surface proteins that possess a semi-rigid and highly extended extracellular domain [1]. P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein [2–4] has recently Received: 12 March 2021 been found to restrict HIV-1 infectivity [5] through steric hindrance [6,7]. Mechanistically, Accepted: 1 May 2021 Published: 4 May 2021 PSGL-1 was found to be incorporated into virion particles, which inhibits virion attachment to target cells [6,7]. The extracellular domain of PSGL-1 was found to be necessary for its

Publisher’s Note: MDPI stays neutral antiviral activity [6]. PSGL-1 shares structural features with other mucins and mucin-like with regard to jurisdictional claims in proteins. It has a heavily glycosylated, elongated extracellular domain that extends nearly published maps and institutional affil- 60 nm from the plasma membrane [8–11] and sterically hinders virus attachment to target iations. cells when incorporated into virions [6,7]. To test whether molecules with a similar structure would also have the ability to block virus infectivity, we selected a group of cellular proteins, including CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123. These proteins have diverse coding sequences, tissue expression patterns, and functionalities, but share a similar Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. structural feature, namely a highly extended and heavily glycosylated extracellular domain. This article is an open access article CD43 is a sialomucin transmembrane expressed at high levels on T lymphocytes, distributed under the terms and , and some B lymphocytes [12]. TIM-1 is preferentially expressed on Th2 T conditions of the Creative Commons cells, and also contains an extracellular mucin domain and an N-terminal immunoglobulin Attribution (CC BY) license (https:// (Ig)-like domain [13]. Both proteins have previously been shown to block HIV-1 infection of creativecommons.org/licenses/by/ T cells [6,7,14]. CD34, the common surface marker of stem and progenitor cells [15], is also a 4.0/). member of the sialomucin family of proteins, which contain an extensively O-glycosylated

Viruses 2021, 13, 832. https://doi.org/10.3390/v13050832 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 832 2 of 15

extracellular mucin-like domain. The podocalyxin-like proteins 1 and 2 (PODXL1 and PODXL2) are CD34-related sialomucins and share a similar structure with CD34 [16], and are also markers of hematopoietic stem and progenitor cells [17,18]. CD164 is another sialomucin co-expressed with CD34 on the surface of human hematopoietic cells, and is structurally similar to CD34 [19]. We also selected two mucins, MUC1 and MUC4, which are transmembrane mucins with dense arrays of O-linked attached to the threonine and serine residues in the extracellular domains [1]. TMEM123 (Porimin) is another mucin-like protein with high contents of glycosylated threonine and serine residues in its extracellular domain [20]. In this study, using HIV-1 infection as a model, we tested these mucins and mucin-like proteins for their ability to inactivate HIV-1 infectivity, and found that all these proteins can block the infectivity of HIV-1 through inhibiting virus particle attachment to target cells. In addition, we also demonstrated that SHREK proteins are broad-spectrum host antiviral factors that block the infection of diverse viruses such as influenza A. These results suggest that SHREK proteins may be a part of host innate immunity against enveloped viruses.

2. Materials and Methods 2.1. Cells and Cell Culture HEK293T (ATCC), MDCK (ATCC), and HeLaJC.53 (kindly provided by Dr. David Kabat) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% heat-inactivated FBS and 50 units/mL of penicillin and 50 µg/mL of streptomycin (Invitrogen). HEK293T(ACE2/TMPRSS2) cells (kindly provided by Virongy) were maintained in DMEM (Invitrogen) supplemented with 10% FBS and puromycin (1 µg/mL) and hygromycin B (200 µg/mL) as instructed by the manufacturer. HIV Rev-dependent GFP indicator Rev-A3R5-GFP cells (kindly provided by Virongy) were cultured in RPMI-1640 plus 10% FBS supplemented with 1 mg/mL geneticin (G418) (Sigma- Aldrich, Saint Louis, MO, USA), 1 µg/mL puromycin (Sigma-Aldrich) and 50 units/mL of penicillin and 50 µg/mL of streptomycin (Invitrogen).

2.2. Plasmid Transfection and Virus Production The infectious HIV-1 molecular clone pHIV-1(NL4-3) was obtained from the NIH AIDS Reagent Program. pCMV3-PSGL-1, pCMV3-CD43, pCMV3-CD164, pCMV3-TMEM123, pCMV3-CD34, pCMV3-PODXL1, pCMV3-PODXL2, pCMV3-TIM1, pCMV3-MUC1, pCMV3-MUC4, pCMV3-Empty, pMUC1-HA, pTMEM123-HA and SARS-CoV-2 S, M, E, or N expression vectors were purchased from Sinobiological. The Ha-CoV-2(Luc) vector was described previously [21]. pCMV6-XL5-ICAM1, pCMV6-XL5-CD2, pCMV6-AC-ITGB2, pCMV6-XL5-CD62L were purchased from Origene. The pHW-NA-GFP(∆AT6) reporter plasmid and the A/WSN/1933 H1N1-derived plasmids pHW2000-PB2, pHW2000-PB1, pHW2000-PA, pHW2000-HA, pHW2000-NP, pHW2000-NA, pHW2000-M, and pHW2000- NS were kindly provided by Dr. Feng Li. For HIV-1 virus production, HEK293T cells were cotransfected in a 6-well plate with 1 µg of pHIV-1(NL4-3) plus the indicated doses of pCMV3-CD164, pCMV3-TMEM123, pCMV3-CD34, pCMV3-PODXL1, pCMV3-PODXL2, pCMV3-TIM1, pCMV3-MUC1, pCMV3-MUC4 or pCMV3-Empty. Supernatants were collected at 48 h post cotransfection. For influenza A-GFP reporter particle assembly, HEK293T cells were cotransfected with pHW2000-PB2, pHW2000-PB1, pHW2000-PA, pHW2000-HA, pHW2000-NP, pHW2000-NA, pHW2000-M, and pHW2000-NS (0.25 µg each); pHW-NA-GFP(∆AT6) (1.5 µg); and pCMV3-PSGL-1, pCMV3-CD43, pCMV3-CD164, pCMV3-TMEM123, pCMV3-CD34, pCMV3-PODXL1, pCMV3-PODXL2, pCMV3-TIM1, pCMV3-MUC1, pCMV3-MUC4, or pCMV3-Empty DNA (0.5 µg each) in a 6-well plate. Viral supernatants were harvested at 48 h. Hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) par- ticles were produced by cotransfection of HEK293T cells in 6-well plates with SARS-CoV-2 S, M, N, and E expression vectors (0.3 µg each), Ha-CoV-2(Luc) (1.2 µg), and each individ- ual SHREK-expressing vector or a control empty vector (1.6 µg). Ha-CoV-2 particles were harvested at 48 h post-transfection. Viruses 2021, 13, 832 3 of 15

2.3. Virus Infectivity Assays For the HIV-1 infectivity assay, p24-normalized HIV-1 particles produced in the pres- ence of PSGL-1, CD43, CD164, TMEM123, CD34, PODXL1, PODXL2, TIM-1, MUC1, MUC4, or empty vector were used to infect Rev-A3R5-GFP cells (0.2 to 0.5 million cells per infec- tion). The percentage of GFP+ cells was quantified by flow cytometry at 48 or 72 h post infection. The IC50 inhibition curves were generated using GraphPad Software. For the influenza A virion infectivity assay, influenza A-GFP reporter viruses were assembled in the presence of PSGL-1, CD43, CD164, TMEM123, CD34, PODXL1, PODXL2, TIM-1, MUC1, MUC4, or empty vectors. Virions were harvested at 48 h and used to infect MDCK cells (3 × 104 cells per infection). GFP expression was quantified at 24 h post infection by flow cytometry. For Ha-CoV-2 infectivity assay, HEK293T(ACE2/TMPRSS2) cells were infected for 2 h with Ha-CoV-2 particles assembled in the presence of each individual SHREK-expressing vector or an empty vector. Infected cells were washed and cultured for 18 h. Cells were lysed with Luciferase Assay Lysis Buffer (Promega, Madison, WI, USA). Luminescence was measured on GloMax® Discover Microplate Reader (Promega).

2.4. HIV Env Incorporation Assay HIV-1 particles were produced by cotransfection of HEK293T cells with pHIV-1(NL4-3) DNA (1 µg) plus 100 ng of vector expressing CD43, CD164, TMEM123, CD34, PODXL1, or PODXL2, or 200 ng of vector expressing MUC1 or MUC4. Empty vector was used to keep the amount of DNA equal in each cotransfection. Virus particles were harvested at 48 h and purified through 10% sucrose gradient by ultracentrifugation (10,000× g for 4 h at 4 ◦C). Particles were resuspended in LDS lysis buffer (Invitrogen) and subjected to Western blot analysis.

2.5. Detection of SHREK Proteins in HIV-1 Particles HIV-1 particles were assembled in 6-well plates by co-transfection of HEK293T cells with pHIV-1(NL4-3) DNA (1 µg) plus an empty vector or the vector expressing each individ- ual SHREK protein (400 ng). For MUC1 and TMEM123, vectors expressing heamagglutinin- tagged MUC1 or TMEM123 were used. Particles were harvested at 48 h, normalized for HIV-1 p24, and subjected to immuno-magnetic capture as previously described [22]. Briefly, magnetic Dynabeads Pan Mouse IgG (Invitrogen) (1 × 108 beads/0.25 mL) were conjugated with one of the following , mouse anti-PSGL-1 (KPL-1) (BD Pharmingen), mouse anti-CD43 antibody (1G10) (BD Biosciences, San Jose, CA, USA), mouse anti-CD164 antibody (67D2) (Biolegend, San Diego, CA, USA), mouse anti-PODXL1 antibody (222328) (R & D Systems, Minneapolis, MN, USA), mouse anti-PODXL2 antibody (R & D Systems), mouse anti-CD34 antibody (563) (BD Biosciences) or mouse anti-HA tag (HA.C5) antibody (Abcam, Cambridge, UK) for 30 min at room temperature. After con- jugation, antibody-conjugated beads were incubated with SHREK-bearing viral particles for 1 h at 37 ◦C. The complex was pulled down with a magnet, and washed with cold PBS for 5 times. Captured viral particles were eluted in 10% Triton x-100 PBS, diluted, and quantified by p24 ELISA.

2.6. Viral Attachment Assay HIV-1 virus particles produced in the presence of PSGL-1, CD43, CD164, TMEM123, CD34, PODXL1, PODXL2, TIM-1, MUC1, MUC4, or empty vector were incubated with HelaJC.53 cells (prechilled at 4 ◦C for 1 h) at 4 ◦C for 2 h. The cells were then washed extensively (5 times) with cold PBS buffer and then lysed with LDS lysis buffer (Invitrogen) for analysis by Western blot. The attachment of HIV-1 virus produced in the presence of TMEM123 was also separately tested by pre-incubating the virus with the mouse mono- clonal anti-TMEM123 antibody (297617) (ThermoFisher, Waltham, MA, USA) (25 µg/mL) at 37 ◦C for 1 h, followed by incubation of the virus with HeLaJC.53 cells at 4 ◦C for 2 h and lysis of the cells with LDS buffer. Viruses 2021, 13, 832 4 of 15

2.7. Western Blots Cells were lysed in LDS lysis buffer (Invitrogen). Proteins were denatured by boiling in sample buffer and subjected to SDS-PAGE, transferred to nitrocellulose membrane, and incubated overnight at 4 ◦C with one of the following primary antibodies: mouse anti-HIV-1 p24 monoclonal antibody (183-H12-5C) (NIH AIDS Reagent Program) (1:1000 dilution), human anti-HIV-1 gp41 antibody (2F5) (1:1000 dilution) (NIH AIDS Reagent Program), mouse anti-MUC1 antibody (HMPV) (BD Biosciences) (1:1000 dilution), mouse monoclonal anti-MUC4 antibody (1G8) (ThermoFisher) (1:1000 dilution), mouse mono- clonal anti-TMEM123 antibody (297617) (ThermoFisher) (1:1000 dilution), or anti-GAPDH goat polyclonal antibody (Abcam) (1:1000 dilution). Membranes were incubated with HRP-labeled goat anti-mouse IgG (KPL) (1:2500 dilution) for 60 min at room temperature, or goat anti-human 800cw-labeled antibodies (Li-Cor Biosciences) (1:2500 dilution) for 30 min at room temperature. Chemiluminescence signal was detected by using West Femto chemiluminescence substrate (Thermo Fisher Scientific), and images were captured with a CCD camera (FluorChem 9900 Imaging Systems) (Alpha Innotech, San Leandro, CA, USA). Blots stained with infrared antibodies were scanned with the Odyssey infrared imager (Li-Cor Biosciences).

2.8. p24 ELISA Detection of extracellular HIV-1 p24 was performed using an in-house p24 ELISA . Briefly, microtiter plates (Sigma-Aldrich) were coated with anti-HIV-1 p24 monoclonal antibody (183-H12-5C) (NIH AIDS Reagent Program). Samples were incubated for 2 h at 37 ◦C, followed by washing and incubating with biotinylated anti-HIV immune globulin (HIVIG) (NIH AIDS Reagent Program) for 1 h at 37 ◦C. Plates were then washed and incubated with -peroxidase conjugate (R &D Systems) for 1 h at 37 ◦C, followed by washing and incubating with tetramethylbenzidine (TMB) substrate. Plates were kinetically read using an ELx808 automatic microplate reader (Bio-Tek Instruments, Winooski, VT, USA) at 630 nm.

2.9. Surface Staining HEK293T cells were transfected with 400 ng of pCMV3-PSGL-1, pCMV3-CD43, pCMV3-CD164, pCMV3-CD34, pCMV3-PODXL1, pCMV3-PODXL2, pCMV3-TIM-1, or pCMV3-Empty DNA. At 48 h post transfection, 0.5–1 million cells were stained with one of the following primary antibodies: mouse anti-PSGL1 antibody (KPL-1) (BD Pharmingen), mouse anti-CD43 antibody (1G10) (BD Biosciences), mouse anti-CD164 antibody (67D2) (Biolegend), mouse anti-CD34 antibody (563) (BD Biosciences), mouse anti-TIM-1 antibody (219211) (R & D Systems), mouse monoclonal anti-PODXL1 antibody (222328) (R & D sys- tems), or mouse monoclonal anti-PODXL2 antibody (211816) (R & D Systems), followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen).

2.10. SARS-CoV-2 S-Antigen ELISA The SARS-CoV2-S antigen kit was purchased from Sinobiological (KIT40591). The ELISA procedure was performed according to the manufacturer’s manual.

3. Results 3.1. Inactivation of HIV-1 Virion Infectivity by Mucins and Mucin-Like Proteins Previous studies have demonstrated that deletion of the highly glycosylated extra- cellular domain of PSGL-1 abolished its antiviral activity [6]. The extracellular domain of PSGL-1 consists of 14–16 decameric repeats (DR) with multiple O-glycosylated threonines (30%) and prolines (10%) [23,24]. DR plays a pivotal role in elongating and strengthening the extracellular N-terminal domain [24]. We found that when the DR domain was deleted from the N-terminus, the anti-HIV activity of PSGL-1 was also abolished (Figure1A,C) [ 6]. These results suggest that the heavily glycosylated, large extracellular domain of PSGL-1 plays a critical role in blocking virion infectivity [6,7]. Viruses 2021, 13, 832 5 of 15 Viruses 2021, 13, x FOR PEER REVIEW 6 of 16

4 A N- or O-linked

MUC1 MUC CD34 PODXL1 PODXL2 TIM-1 CD43 TMEM123 CD164 PSGL-1

S PSGL-1∆R S S

S S S S S S S

COOH COOH COOH COOH COOH COOH COOH COOH COOH

PSGL-1 CD43 TIM-1 B SHREK CD34 HIV-1 HIV-1 PODXL1 ITGB-2 PODXL2 CD164 E-Selectin MUC1 ICAM-1 MUC4 L-Selectin TMEM123

CD2, ICAM-1, L-Selectin, CD2 E-Selectin, ITGB-2 Ig domain consensus repeats consensus EGF repeats

COOH COOH COOH COOH COOH C Rev-A3R5-GFP Rev-A3R5-GFP Rev-A3R5-GFP + HIV + HIV + HIV Indicator cells Indicator cells Cell only (vector) (PSGL-1) (PSGL-1ΔDR) 4 4 4 4 10 10 10 10

3 3 3 3 10 0.07% 10 28.03% 10 0.11% 10 24.02%

2 2 2 2 10 10 10 10 PI 1 1 1 1 10 10 10 10

0 0 0 0 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

D GFP

Rev-A3R5-GFP + HIV + HIV + HIV + HIV + HIV Cell only (Vector) (PSGL-1) (CD43) (TIM-1) (CD34) 4 4 4 4 4 4 10 10 10 10 10 10

3 3 3 3 3 3 10 10 10 10 10 10 0.1% 3.3% 0.1% 0.1% 0.1% 0.1% 2 2 2 2 2 2 10 10 10 10 10 10

1 1 1 1 1 1 10 10 10 10 10 10

0 0 0 0 0 0 10 10 10 10 10 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 + HIV + HIV + HIV + HIV + HIV + HIV (PODXL1) (PODXL2) (CD164) (MUC1) (MUC4) (TMEM123) 4 4 4 4 4 4 10 10 10 10 10 10

3 3 3 3 3 3 10 10 10 10 10 10 0.3% 0% 0.1% 0.8% 0.6% 0.1% 2 2 2 2 2 2 10 10 10 10 10 10

1 1 1 1 1 1 PI 10 10 10 10 10 10

100 100 100 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

GFP E Rev-A3R5-GFP + HIV + HIV + HIV + HIV + HIV Cell only (vector) (CD2) (ICAM-1) (L-selectin) (ITGB2) 4 4 4 4 4 4 10 10 10 10 10 10

3 3 3 3 3 3 10 0.08% 10 9.86% 10 21.95% 10 8.28% 10 11.08% 10 7.33%

2 2 2 2 2 2 10 10 10 10 10 10

1 1 1 1 1 1 10 10 10 10 10 10 PI

100 100 100 100 100 100 0 1 2 34 0 1 2 34 0 1 2 34 0 1 2 34 0 1 2 34 0 1 2 34 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

GFP Figure 1. (A) SchematicFigure of 1. PSGL-1(A) Schematic and PSGL-1 of∆ PSGL-1DR mutant, and SHREK PSGL-1 proteins,∆DR mutant,and other SHREKsurface receptors proteins, used and in otherthis surface study. (B) Schematic of virion production in the presence of SHREK proteins in virus producer cells. (C) Requirement of B the DR domain ofreceptors PSGL-1 for used blocking in this HIV-1 study. infectivity. ( ) Schematic HEK293T of cells virion were production cotransfected in with the HIV(NL4-3) presence of DNA SHREK (1 µg) proteins in plus the vector expressingvirus producer PSGL-1 or cells. PSGL-1 (C)∆ RequirementDR (400 ng). Virions of the were DR harvested domain at of 48 PSGL-1 h post-transfection for blocking and HIV-1normal- infectivity. ized for p24. ViralHEK293T infectivity cells was werequantified cotransfected by infecting with Rev-A3R5-GFP HIV(NL4-3) indicator DNA (1cellsµg) with plus 300 the ng vector p24 input expressing of the vi- PSGL-1 or ruses. HIV-1 replication was quantified by GFP expression at 72 h post infection. This experiment was repeated 3 times. PSGL-1∆DR (400 ng). Virions were harvested at 48 h post-transfection and normalized for p24. Viral (D) SHREK proteins inactivate HIV-1 virion infectivity. HEK293T cells were cotransfected with HIV(NL4-3) DNA (1 µg) plus each individualinfectivity SHREK-expressing was quantified vector (100 by infectingng for TIM-1, Rev-A3R5-GFP 500 ng for other indicatorSHREKs). Virions cells with were300 harvested ng p24 at 48 input of the h and normalizedviruses. for p24, HIV-1and viral replication infectivity was quantified quantified by byinfecting GFP expressionRev-A3R5-GFP at 72indicator h post cells infection. with 35 Thisng p24 experiment input of each virus.was Shown repeated are the 3 times.percentages (D) of SHREK GFP+ cells proteins at 72 h inactivatepost infection. HIV-1 The experiment virion infectivity. was repeated HEK293T 3 times. cells were (E) For comparison, cells were also similarly cotransfected with HIV-1(NL4-3) DNA plus an empty vector or the vector cotransfected with HIV(NL4-3) DNA (1 µg) plus each individual SHREK-expressing vector (100 ng for TIM-1, 500 ng for other SHREKs). Virions were harvested at 48 h and normalized for p24, and viral infectivity was quantified by infecting Rev-A3R5-GFP indicator cells with 35 ng p24 input of each virus. Shown are the percentages of GFP+ cells at 72 h post infection. The experiment was repeated 3 times. (E) For comparison, cells were also similarly cotransfected with HIV-1(NL4-3) DNA plus an empty vector or the vector expressing CD2, ICAM-1, L-selectin, or ITGB2. Virions were harvested and quantified by infecting Rev-A3R5-GFP indicator cells with 50 ng p24 input of each virus. The experiment was repeated 3 times. Viruses 2021, 13, 832 6 of 15

To determine whether mucins and mucin-like proteins with a similar extracellular structure would also inhibit HIV-1 virion infectivity, we selected and tested a group of mucins and mucin-like proteins, including CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123 (Figure1A). We cotransfected them individually with HIV- 1(NL4-3) DNA into HEK293T cells to assemble viral particles, and then quantified the infectivity of the virions produced by infecting an HIV-1 Rev-dependent indicator cell, Rev- A3R5-GFP [6,25,26] (using an equal level of p24). The presence of each of these proteins in virus producer cells blocked the infectivity of the HIV-1 virions (Figure1D). To determine whether transmembrane proteins with large extracellular domains would similarly inhibit HIV-1 infectivity, we selected several non-mucin proteins with various extracellular repeat domains. Both CD2 and ICAM-1 are members of the im- munoglobulin (Ig) superfamily of proteins that contain heavily glycosylated extracellular Ig domains (2 and 5 Ig domain repeats for CD2 and ICAM-1, respectively) [27]. ICAM-1 has been shown to enhance virion infectivity in an ICAM-1/LFA-1-dependent fashion [28–31]. L-selectin and E-selectin are members of the selectin family of molecules that share a similar extracellular structure with a variable number of consensus repeats (2 and 6 for L- and E-, respectively) [32]. We also selected beta chain-2 (ITGB2), which has a large extracellular domain with integrin epidermal growth factor like repeat domains (I-EGF repeats 1 to 4) [33]. Each of these proteins was similarly cotransfected with HIV-1(NL4-3) DNA into HEK293T cells to assemble viral particles. Virions released were harvested and quantified for infectivity in Rev-A3R5-GFP cells (using an equal level of p24). As shown in Figure1E, none of these proteins, except for E-selectin (Supplementary Figure S1), was found to block the infectivity of HIV-1 virions. This is in contrast with the mucin and mucin-like proteins that we selected; all of these mucin and mucin-like proteins were found to block HIV-1 infectivity (Figure1D). These results demonstrate that the ability of mucin and mucin-like proteins to block HIV-1 infectivity is not a shared property of transmembrane proteins expressed in virus producer cells. We further performed experiments to quantify and compare the anti-HIV activity of the mucin and mucin-like proteins (Supplementary Figures S2–S4). We observed dosage- dependent inhibition of HIV-1 by all of them (Figure2A,B, Supplementary Figures S5–S11), and the 50% inhibition dosages were determined (Figure2C). Among these proteins, CD34, the common maker, had the strongest inhibition, blocking HIV-1 virion infectivity at an IC50 of 2.33 ng (Figure2C). The MUC1 vector was less effective, with an IC 50 of 216.06 ng (Figure2C). Based on the antiviral activities and shared structural feature of these proteins, we conveniently named them the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators. Viruses 2021, 13, x FOR PEER REVIEW 7 of 16

expressing CD2, ICAM-1, L-selectin, or ITGB2. Virions were harvested and quantified by infecting Rev-A3R5-GFP indica- tor cells with 50 ng p24 input of each virus. The experiment was repeated 3 times.

We further performed experiments to quantify and compare the anti-HIV activity of the mucin and mucin-like proteins (Supplementary Figures S2–S4). We observed dosage- dependent inhibition of HIV-1 by all of them (Figure 2A,B, Supplementary Figures S5– S11), and the 50% inhibition dosages were determined (Figure 2C). Among these proteins, CD34, the common stem cell maker, had the strongest inhibition, blocking HIV-1 virion infectivity at an IC50 of 2.33 ng (Figure 2C). The MUC1 vector was less effective, with an Viruses 2021, 13, 832 IC50 of 216.06 ng (Figure 2C). Based on the antiviral activities and shared structural feature7 of 15 of these proteins, we conveniently named them the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators.

A Rev-A3R5-GFP + HIV + HIV + HIV + HIV C Cell only (PODXL2, 0 ng ) (PODXL2, 0.5 ng ) (PODXL2, 1 ng ) (PODXL2, 5 ng ) 4 4 4 4 4 10 10 10 10 10 SHREK M.W. Half Maximal Inhibition 3 3 3 3 3 10 10 10 10 10 Proteins (KD) Dosage (IC ) (ng) 0.1% 10.0% 6.8% 4.5% 4.3% 50 2 2 2 2 2 10 10 10 10 10 CD3490 - 120 2.33 1 1 1 1 1 10 10 10 10 10 CD43115 - 130 2.69 0 0 0 0 0 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 PSGL-1120 2.70 PODXL2120 - 160 2.81 + HIV + HIV + HIV + HIV + HIV PODXL1160 - 170 4.33 (PODXL2, 10 ng ) (PODXL2, 50 ng ) (PODXL2, 100 ng ) (PODXL2, 200 ng ) (PODXL2, 400 ng ) 4 4 4 4 4 10 10 10 10 10 CD16480 - 100 5.38 TIM-170 - 80 7.98 3 3 3 3 3 10 10 10 10 10 3.4% 1.7% 0.8% 0.4% 0.2% TEME123110 11.44 2 2 2 2 2 10 10 10 10 10 MUC4550 - 930 12.53 PI 1 1 1 1 1 10 10 10 10 10 MUC1> 200 216.06

0 0 0 0 0 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 GFP B

150 IC50 = 7.98 ng IC50 = 2.34 ngIC50 = 4.33 ng IC50 = 2.81 ng IC50 = 5.38 ng IC50 = 216.06 ng IC50 = 12.53 ng IC50 = 11.44 ng

100

50

0 01234560123456012345601234560123456012345601234560123456 % Relative Infectivity % Relative Log (TIM-1, pg) Log (CD34, pg)Log (PODXL1, pg) Log (PODXL2, pg) Log (CD164, pg) Log (MUC1, pg) Log (MUC4, pg) Log (TMEM123, pg) 10 10 10 10 10 10 10 10 Figure 2.2.( A(A) Dose-dependent) Dose-dependent inhibition inhibition of HIV-1 of HIV-1 infectivity infectivity by PODXL2. by PODXL2. HEK293T HEK293T cells were cotransfectedcells were cotransfected with pHIV(NL4-3) with DNApHIV(NL4-3) (1 µg) plus DNA the (1 PODXL2 µg) plus expression the PODXL2 vector expression (0.5 to 400 vector ng of (0.5 DNA). to 400 Virions ng of DNA). were harvested Virions were at 48 harvested h and normalized at 48 h and for p24,normalized and viral for infectivity p24, and wasviral quantified infectivity by was infecting quanti Rev-A3R5-GFPfied by infecting indicator Rev-A3R5-GFP cells. (B) indicator Virions were cells. assembled (B) Virions in were the pres- as- encesembled of each in the individual presence SHERK of each protein, individual and quantified SHERK protein, for virus and infectivity quantifi ased in for (A ).virus The dose-dependentinfectivity as in inhibition(A). The dose-de- of HIV-1 pendent inhibition of HIV-1 curve by each individual SHREK protein was plotted using results from 2 independent ex- curve by each individual SHREK protein was plotted using results from 2 independent experiments. (C) The 50% inhibition periments. (C) The 50% inhibition dosage (IC50) for each SHREK protein was calculated based on the averages from the dosage (IC ) for each SHREK protein was calculated based on the averages from the two independent experiments. two independent50 experiments.

3.2. Effects Effects of SHREK Protein Expression on HIV-1 Virion Release To elucidate the the anti-HIV anti-HIV mechanisms mechanisms of of SHREK SHREK proteins, proteins, we we first first examined examined the theef- fectseffects of of SHREK SHREK expression expression on on virion virion release release in in virus virus producer producer cells. Previous studies of PSGL-1 have demonstrated that expression of PSGL-1 in virus producer cells had minimal effects on HIV-1 virion release [[6].6]. To examine effects of SHREK proteins on HIV-1 virion µ release, we cotransfected HEK293T cells with HIV(NL4-3) proviral DNA (1 µg)g) plus each of the SHREK protein expression vector vector at at va varyingrying inputs inputs from from 0.5 0.5 to to 400 400 ng ng (Figure (Figure 33).). The release of virions in the presence of eacheach SHREKSHREK proteinprotein waswas quantified.quantified. All of the proteins exceptexcept TIM-1TIM-1 hadhad minorminor effects effects on on virion virion release release at at doses doses below below 100 100 ng ng (Figure (Figure3 ). CD34, PODXL1, and CD164 [34] inhibited HIV-1 virion release only at high dosages 3). CD34, PODXL1, and CD164 [34] inhibited HIV-1 virion release only at high dosages (100 ng and above). TIM-1 potently inhibited virion release at doses as low as 5–10 ng (100 ng and above). TIM-1 potently inhibited virion release at doses as low as 5–10 ng (Figure3 and Supplementary Figure S12), consistent with a previous report [ 14]. While (Figure 3 and Supplementary Figure S12), consistent with a previous report [14]. While not inhibiting virion release at low dosages, all of these proteins, except for MUC1 (IC50, not inhibiting virion release at low dosages, all of these proteins, except for MUC1 (IC50, 216.05), effectively blocked virion infectivity with an IC50 at approximately 10 ng and below 216.05), effectively blocked virion infectivity with an IC50 at approximately 10 ng and (Figure2B,C). These results suggested that, for most of these SHREK proteins, inactivating virion infectivity rather than blocking virion release is a major mechanism.

Viruses 2021, 13, x FOR PEER REVIEW 8 of 16

Viruses 2021, 13, 832 8 of 15 below (Figure 2B,C). These results suggested that, for most of these SHREK proteins, in- activating virion infectivity rather than blocking virion release is a major mechanism.

FigureFigure 3. 3.Effects Effects of of SHREK SHREK proteinsproteins on HIV-1 HIV-1 viral viral release. release. HEK HEK293T293T cells cells were were cotransfected cotransfected with withHIV(NL4-3) HIV(NL4-3) DNA DNA(1 µg) plus each individual SHREK expression vector (0.5 to 400 ng of DNA). Virion release was quantified at 48 h post (1 µg) plus each individual SHREK expression vector (0.5 to 400 ng of DNA). Virion release was quantified at 48 h post cotransfection by HIV-1 p24 ELISA. Data are represented as mean ± SD from ELISA triplicate. Experiments on the effects cotransfectionof TIM-1 were by HIV-1independently p24 ELISA. repeated Data are3 times represented (Figure asS12) mean, and± experimentsSD from ELISA on effects triplicate. of other Experiments SHREKs on were the effectsinde- of TIM-1pendently were independentlyrepeated twice. repeated 3 times (Figure S12), and experiments on effects of other SHREKs were independently repeated twice. 3.3. Virion Incorporation of SHREK Proteins and Effects on HIV-1 Env Incorporation 3.3. Virion Incorporation of SHREK Proteins and Effects on HIV-1 Env Incorporation The anti-HIV activity of PSGL-1 is mainly attributed to its incorporation into virion particlesThe anti-HIVthat sterically activity hinders of PSGL-1 virion attachment is mainly attributed to target cells to its [6,7]. incorporation In addition, into virion virion particlesincorporation thatsterically of PSGL-1 hinders also inhibits virion the attachment incorporation to target of the cells HIV-1 [6, 7Env]. In protein addition, [6,35]. virion incorporationTo study the ofanti-viral PSGL-1 mechanisms also inhibits theof SHREK, incorporation we examined of the HIV-1 virion Env incorporation protein [6,35 of]. To studySHERK the proteins. anti-viral For mechanisms this purpose, of SHREK,we adopted we examineda previously virion established incorporation particle of pull- SHERK proteins.down assay For [22], this using purpose, magnetic we adopted beads-conjug a previouslyated anti-SHREK established antibodies particle to pull-downpull down as- sayvirion [22 ],particles using magneticthat express beads-conjugated SHREK on the su anti-SHREKrface (Figure 4A). antibodies Magnetically to pull purified down virionvi- particlesrions were that quantified express for SHREK the presence on the of surface HIV-1 p24 (Figure in the4A). virions. Magnetically We were able purified to demon- virions werestrate quantified that the anti-PSGL-1 for the presence antibody of HIV-1 select p24ively in pulled the virions. down Wep24+ were virion able particles to demonstrate pro- thatduced the from anti-PSGL-1 the PSGL-1 antibody expression selectively cells, but pulled not from down the p24+ cells virionco-transfected particles with produced the fromempty the vector, PSGL-1 recapitulating expression previous cells, but findings not from that the PSGL-1 cells co-transfected is incorporated with into the HIV-1 empty vector,virion particles recapitulating [5–7]. Using previous this pull-down findings that assay, PSGL-1 we were is incorporatedable to demonstrate into HIV-1 that sim- virion particlesilar to PSGL-1, [5–7]. Usingthe anti-CD43, this pull-down -CD164, assay,-PODXL1, we were-PODXL2, able toand demonstrate -CD34 antibodies that similar selec- to PSGL-1,tively pulled the anti-CD43, down virion -CD164, particles -PODXL1, produced -PODXL2,from these SHREK and -CD34 expression antibodies cells selectively(Figure pulled4A,B), downdemonstrating virion particles virion incorporation produced from of SHREK these SHREK proteins. expression The anti-TMEM123 cells (Figure anti-4A,B), demonstratingbody had a weak virion ability incorporation to pull down of virion SHREK particles proteins. produced The anti-TMEM123 from the TMEM123 antibody ex- had apression weak ability cells. toWe pull were down not virionable to particlesuse the commercial produced fromanti-MUC1 the TMEM123 and anti-MUC4 expression anti- cells. Webodies were to notpull abledown to virion use the particles. commercial To overcome anti-MUC1 these tec andhnique anti-MUC4 difficulties, antibodies we replaced to pull downTMEM123 virion and particles. MUC1 with To overcome hemagglutinin-tagge these techniqued TMEM123 difficulties, or MUC1 we replacedto assemble TMEM123 viral and MUC1 with hemagglutinin-tagged TMEM123 or MUC1 to assemble viral particles, and then used magnetic beads-conjugated anti-hemagglutinin antibody to pull down virion particles. Using this approach, we were able to demonstrate virion incorporation of TMEM123 and MUC1 (Figure4A,B). Viruses 2021, 13, x FOR PEER REVIEW 9 of 16

particles, and then used magnetic beads-conjugated anti-hemagglutinin antibody to pull Viruses 2021, 13, 832 down virion particles. Using this approach, we were able to demonstrate virion incorpo- 9 of 15 ration of TMEM123 and MUC1 (Figure 4A,B).

Figure 4. Virion incorporation of SHREK proteins and its effects on HIV-1 Env incorporation. (A) Schematic of the immune- Figure 4. Virion incorporation of SHREK proteins and its effects on HIV-1 Env incorporation. (A) Schematic of the im- magneticmune-magnetic capture assay capture used assay to detect used to SHREK detect SHREK proteins proteins on HIV-1 on HIV-1 particles. particles. (B) ( HEK293TB) HEK293T cells cells were were cotransfectedcotransfected with pHIV-1(NL4-3)with pHIV-1(NL4-3) DNA (1 µg) DNA plus (1 eachµg) plus individual each individual SHREK SHREK protein protein expression expression vector vector or or empty empty vector vector (400 ng ng of of DNA). DNA). As a control,As cells a control, were alsocells were transfected also transfected with only with SHREK-expressing only SHREK-expressing vector vector (400 (400 ng). ng). Empty Empty vector was was used used to maintain to maintain an an equal DNA concentration. For MUC1 and TMEM123, vectors expressing hemagglutinin-tagged TMEM123 or MUC1 equal DNAwere concentration.used. Supernatants For were MUC1 harvested and TMEM123, at 48 h, normalized vectors expressing for p24, and hemagglutinin-tagged incubated with magnetic TMEM123 beads coated or MUC1 with were used. Supernatantsantibodies forwere each individual harvested SHREK at 48 h, or normalized hemagglutinin. for p24,Captured and incubatedparticles were with washed, magnetic eluted, beads and quantified coated with for antibodiesthe for eachp24 individual levels with SHREK HIV-1 orp24 hemagglutinin. ELISA. Data are Capturedpresented as particles the percentage were washed, of input eluted,particles and captured quantified by the forbeads. the The p24 levels with HIV-1experiment p24 ELISA. was independently Data are presented repeated 3 as times, the percentageand the means of ± input SD from particles experiment captured triplicate by are the shown. beads. (C The) Effect experiment of SHREK proteins on HIV-1 Env incorporation. HEK293T cells were cotransfected with HIV(NL4-3) DNA plus each indi- was independentlyvidual SHREK repeated protein expression 3 times, andvector the or meansan empty± vector.SD from Particles experiment were harv triplicateested at are48 h, shown. and purified (C) Effect through of a SHREK proteinssucrose on HIV-1 gradient. Env Virions incorporation. were lysedHEK293T and analyzed cells with were Western cotransfected blot using anti withbodies HIV(NL4-3) against HIV-1 DNA gp41 plus and eachp24. Rep- individual SHREKresentative protein expression blots from 3 vector independent or an experiment empty vector. repeats Particles are shown. were The harvested band intensities at 48 h,were and quantified purified from through the three a sucrose blots and normalized for p24. p-values were calculated using the two-tailed T-test. Significance values are indicated using gradient. Virions were lysed and analyzed with Western blot using antibodies against HIV-1 gp41 and p24. Representative asterisks as follows; * = p < 0.05, *** = p < 0.001, **** = p < 0.0001. blots from 3 independent experiment repeats are shown. The band intensities were quantified from the three blots and normalized for p24. p-values were calculatedWe further using examined the two-tailed effects T-test.of SHREK Significance incorporation values on are HIV-1 indicated Env usingincorporation. asterisks as follows; * = p < 0.05, *** = p < 0.001,Virion **** particles = p < 0.0001. were assembled in the presence or absence of SHREK proteins, and then analyzed by western blots for HIV-1 Env gp41 and p24. As shown in Figure 4C, all We further examined effects of SHREK incorporation on HIV-1 Env incorporation. Virion particles were assembled in the presence or absence of SHREK proteins, and then analyzed by western blots for HIV-1 Env gp41 and p24. As shown in Figure4C, all SHREKs, except for TMEM123, had low-level inhibition of HIV-1 Env incorporation to various degrees; while TMEM123 did not affect Env incorporation, PODXL2 had the strongest inhibition.

3.4. SHREK Proteins Inhibit Virus Particle Attachment to Target Cells We and others previously reported that PSGL-1-mediated inhibition of virion in- fectivity occurs mainly through virion incorporation, leading to PSGL-1-mediated steric Viruses 2021, 13, x FOR PEER REVIEW 10 of 16

SHREKs, except for TMEM123, had low-level inhibition of HIV-1 Env incorporation to various degrees; while TMEM123 did not affect Env incorporation, PODXL2 had the Viruses 2021, 13, 832 strongest inhibition. 10 of 15

3.4. SHREK Proteins Inhibit Virus Particle Attachment to Target Cells We and others previously reported that PSGL-1-mediated inhibition of virion infec- hindrancetivity occurs of particle mainly through attachment virion to incorporation, target cells [6 ,leading7]. We performedto PSGL-1-mediated a similar steric virion hin- attach- mentdrance assay of usingparticle particles attachment produced to target from cells cells [6,7]. expressing We performed each individuala similar virion SHREK attach- protein. Asment shown assay in Figureusing particles5A, as a produced control, particles from cells produced expressing from each the individual PSGL-1-expressing SHREK pro- cells weretein. highly As shown impaired in Figure in 5A, their as ability a control, to attachparticles to produced target cells. from Similar the PSGL-1-expressing strong impairment wascells observed were highly for virions impaired produced in their ability from theto attach CD43-, to CD34-,target cells. and Similar PODXL2-expressing strong impair- cells. Ofment note, was these observed four proteins for virions (PSGL-1, produced CD43, from CD34, the CD43-, and PODXL2)CD34-, and have PODXL2-expressing the strongest inhibi- tioncells. of Of HIV-1 note, among these four SHREKs, proteins with (PSGL-1, an IC CD43,50 around CD34, 2 and ng (FigurePODXL2)2C). have The the other strongest proteins (CD164,inhibition PODXL1, of HIV-1 MUC1, among and SHREKs, MUC4) with also an inhibited IC50 around virion 2 ng attachment(Figure 2C). toThe varying other pro- degrees. Interestingly,teins (CD164, TMEM123 PODXL1, didMUC1, not significantlyand MUC4) also inhibit inhibited HIV-1 virion virion attachment attachment to to varying target cells, althoughdegrees. itInterestingly, inhibited viral TMEM123 infection did (Figure not signif2B,C).icantly One inhibit possible HIV-1 reason virion isattachment that TMEM123 to itselftarget may cells, interact although with it inhibited surface proteins, viral infection thereby (Figure promoting 2B,C). One non-productive possible reason attachment is that ofTMEM123 virus particles itself may to target interact cells. with Indeed, surface whenproteins, the thereby binding promoting assay was non-productive performed in the attachment of virus particles to target cells. Indeed, when the binding assay was per- presence of an anti-TMEM123 antibody to block possible TMEM123 interaction with cell formed in the presence of an anti-TMEM123 antibody to block possible TMEM123 inter- surface proteins, virion attachment was decreased (Figure5B). action with cell surface proteins, virion attachment was decreased (Figure 5B).

− α-TMEM123 + α-TMEM123 A 3 B 2 1 2 L r -1 4 X 1 4 to GL 43 16 D DXL D D S MEM1 O O r r Vec P C C T CD34 P P MUC MUC o M123 o ct E ct EM123 e M Cell-associated V TM Ve T p24 Virion

Cell-associated p24 Virion GAPDH GAPDH

0.7 1.8 * 0.6 1.6 1.4 0.5 * * * 1.2 0.4 1.0 * 0.3 0.8

p24/GAPDH Intensity p24/GAPDH *** *** 0.6 *** p24/GAPDHIntensity 0.2 *** 0.4 0.1 0.2 0 0 r r o 3 or -1 3 4 4 2 1 4 t 2 t L 4 16 3 3 X L C C c cto ec G D D 12 D D X U U e V S C C M C O D M M Ve M1 V P E P PO E TM M T TMEM123 − α-TMEM123 + α-TMEM123 FigureFigure 5. SHREK 5. SHREK proteins proteins block block HIV-1 HIV-1 virion virion attachment attachment to to target target cells. cells. ((AA)) ViralViral particlesparticles were were pr producedoduced by by cotransfec- cotransfection of HEK293Ttion of HEK293T cells with cells pHIV-1(NL4-3) with pHIV-1(NL4-3) DNA DNA (1 µ (1g) µg) and and each each individual individual SHREKSHREK protein protein expr expressionession vector vector or an or empty an empty vector (500 ng). HIV-1 p24-normalized viral particles were then assayed for attachment to target HeLaJC.53 cells by West- vector (500 ng). HIV-1 p24-normalized viral particles were then assayed for attachment to target HeLaJC.53 cells by Western ern blot for cell-bound p24. (B) Virions produced in the presence of the TMEM123 expression vector or the empty vector blot forwere cell-bound assayed for p24. attachment (B) Virions in the produced presence inor theabsence presence of an ofanti-TMEM123 the TMEM123 antibody. expression Repres vectorentative or theblots empty from 3 vector exper- were assayediment for repeats attachment are shown. in the The presence band intensities or absence were ofan quantified anti-TMEM123 (for A and antibody. B) from Representativethe three blots and blots normalized from 3 experiment with repeatsGAPDH. are shown. p-values The were band calculated intensities using werethe two-tailed quantified T-test. (for SignifA,B)icance from values the three are indicated blots and using normalized asterisks as with follows; GAPDH. p-values* = p were< 0.05, calculated *** = p < 0.001. using the two-tailed T-test. Significance values are indicated using asterisks as follows; * = p < 0.05, *** = p < 0.001. 3.5. SHREK Proteins Are Broad-Spectrum Host Antiviral Factors 3.5. SHREKPSGL-1 Proteins has been Are shown Broad-Spectrum to be a broad-spectrum Host Antiviral host Factors antiviral factors [6]. To further determinePSGL-1 whether has been these shown SHREK to beproteins a broad-spectrum also possess broad-spectrum host antiviral antiviral factors [activities,6]. To further determinewe tested whether their ability these to SHREK block other proteins viruses, also including possess broad-spectruminfluenza A and antivirala new hybrid activities, alphavirus-SARS-CoV-2 pseudovirus [21]. To assemble influenza A virus, eight vectors we tested their ability to block other viruses, including influenza A and a new hybrid alphavirus-SARS-CoV-2 pseudovirus [21]. To assemble influenza A virus, eight vectors expressing each of the segments of the influenza A/WSN/33 (H1N1) genome plus a GFP-reporter vector were cotransfected with individual SHREK proteins into HEK293T (Figure6A ). Viral particles were harvested and used to infect target MDCK cells. As shown in Figure6B, the presence of each of the SHREK proteins in virus-producer cells inhibited the infection of target cells by the virions released. However, the degree of inhibition was different. MUC1, PODXL1, and MUC4 had the strongest inhibition of influenza A virus, although MUC1 and MUC4 were less effective against HIV-1. CD164 and TMEM123 had the weakest inhibition of influenza A. These results demonstrate that SHREK proteins Viruses 2021, 13, 832 11 of 15

are broad-spectrum and can block the infectivity of multiple viruses. Nevertheless, it is Viruses 2021, 13, x FOR PEER REVIEW apparent that for each individual SHREK, its antiviral potency can vary among12 of different16 viruses, with the differences likely related to possible viral antagonisms, the localization of SHREKs and sites of viral budding, and other unidentified factors.

Figure 6. A Figure 6. SHREKSHREK proteins proteins are broad-spectrum are broad-spectrum host hostantiviral antiviral factors. factors. (A) SHREK ( ) SHREK proteins proteins inacti- inactivate vateinfluenza influenza A A virion virion infectivity. infectivity. Influenza Influenza A-GFP A-GFP reporterreporter virusesviruses were assembled by by cotrans- cotransfection of fectionHEK293T of HEK293T cells with cells eight with vectorseight vectors expressing expressing each each of the of segments the segments of influenza of influenza A/WSN/33 (H1N1) A/WSN/33(0.25 µg (H1N1) each), pHW-NA-GFP(0.25 µg each), pHW-NA-GFP (∆AT6) (1.5 µ g),(ΔAT6) and (1.5 each µg), individual and each SHREK-expressingindividual SHREK- vector or expressingan empty vector vector or an (0.5 emptyµg). Viralvector particles (0.5 µg). Viral were particles harvested were at 48harvested h and usedat 48 h to and infect used target to MDCK infect target MDCK cells. This experiment was repeated 3 times. (B) Representative results from cells. This experiment was repeated 3 times. (B) Representative results from (A), showing GFP+ (A), showing GFP+ cells being quantified by flow cytometry following infection for 24 h. (C) SHREKcells beingproteins quantified inhibit Ha-CoV-2 by flow infection. cytometry Ha-C followingoV-2(Luc) infection particles for were 24 h. assembled (C) SHREK in HEK293T proteins inhibit cellsHa-CoV-2 by cotransfection infection. of Ha-CoV-2(Luc)SARS-CoV-2 S-, particlesM-, N- and were E- expressing assembled vectors in HEK293T (0.3 µg each), cells by Ha-CoV- cotransfection 2(Luc)of SARS-CoV-2 vector (1.2 µg), S-, and M-, each N- and individual E- expressing SHREK- vectorsexpressing (0.3 vectorµg each), or an Ha-CoV-2(Luc) empty vector (1.6 vector µg). (1.2 µg), Virionsand eachwere individual harvested, SHREK-expressingand used to infect HE vectorK293T(ACE2/TMPRSS2) or an empty vector target (1.6 µ cells.g). Virions Luciferase were ac- harvested, tivityand was used quantified to infect at HEK293T(ACE2/TMPRSS2) 18 h post infection. The experiment target cells. was Luciferaserepeated four activity times. was Data quantified are pre- at 18 h sented as means ± SD from experiment triplicate. p-values were calculated using the two-tailed T- post infection. The experiment was repeated four times. Data are presented as means ± SD from test. Significance values are indicated using asterisks as follows; **** = p < 0.0001. experiment triplicate. p-values were calculated using the two-tailed T-test. Significance values are 4. Discussionindicated using asterisks as follows; **** = p < 0.0001. In this report, we identified a group of proteins, namely SHREK, that share similar structural characteristics and the ability to broadly inactivate virion infectivity (Figure 1).

Viruses 2021, 13, 832 12 of 15

To test possible effects of SHREK on SARS-CoV-2 infection, we assembled a newly developed hybrid alphavirus-SARS-CoV-2 particles (Ha-CoV-2) [21] in the presence of individual SHREKs, using PSGL-1 as a control. Ha-CoV-2 is a non-replicating SARS-CoV-2 virus-like particle, composed of SARS-CoV-2 structural proteins (S, M, N, and E) and an RNA genome derived from an alphavirus vector. Our recent study has shown that PSGL-1 can block SARS-CoV-2 S protein virion incorporation, virus attachment, and Ha-CoV-2 infection of target cells [36]. We performed similar experiments on the inhibition of Ha- CoV-2 infection by SHREKs, and found that in addition to PSGL-1, CD164, TIM-1, MUC1, and MUC4 also inhibited the infection of Ha-CoV-2 virus (Figure6C); The MUC4 vector had the strongest inhibition among these SHREKs. However, we did not find inhibition of Ha-CoV-2 by CD43, CD34, PODXL1, PODXL2, and TMEM123 (Figure S13), although they are potent inhibitors of HIV-1 (Figure2B,C). We further examined possible effects of SHREK proteins on Ha-CoV-2 S protein incorporation and virion release by quantifying the amounts of S in the supernatant (Sup- plementary Figure S14). Expression of MUC4 greatly reduced the S protein concentration, likely through blocking virion release or S incorporation; expression of MUC1 or PSGL-1 also led to a roughly 50% reduction of S in the supernatant [36]. For the rest of SHREK proteins, their expression had minor effects on virion release or S protein incorporation.

4. Discussion In this report, we identified a group of proteins, namely SHREK, that share similar structural characteristics and the ability to broadly inactivate virion infectivity (Figure1 ). We also quantified their antiviral activity in a range of doses (from 0.5 ng to 400 ng of SHREK expression vector), using HIV-1 infection as a model, and observed a strictly dosage- dependent inhibition of HIV-1 virion infectivity (Figure2). The physiological relevance of SHREK proteins such as PSGL-1 in restricting HIV-1 virion infectivity has been previously confirmed in HIV-1 infection of primary CD4 T cells, and by shRNA knockdown of PSGL-1 in human CD4 T cells [6]. The slight reduction of endogenous levels of PSGL- 1 in transformed and primary CD4 T cells led to an enhancement of virion infectivity, demonstrating that the presence of endogenous PSGL-1 can affect virion infectivity [6]. Although we mainly focused on proteins with mucin and mucin-like domains, we ex- pect that SHREK proteins will likely include other proteins, such as E-selectin (Figure S1B), with diverse molecular structures. The antiviral activities of SHREK can be achieved through at least three different mechanisms: (1) blocking virion release, e.g., TIM-1 inhibi- tion of HIV-1 release [14]; (2) inhibition of virion incorporation of viral attachment proteins, e.g., PSGL-1 inhibition of HIV-1 gp160 incorporation [6]; (3) virion incorporation of SHREK that blocks progeny virus attachment to target cells through steric hindrance (e.g., PSGL-1 inhibition of HIV-1 virus attachment to target cells [6,7]). Our previous studies have also shown that viruses such as HIV-1 can evolve antagonisms to counteract the blockage imposed by SHREK proteins. For example, HIV-1 uses the accessory proteins Vpu and Nef to degrade and downregulate PSGL-1 on CD4 T cells [5,6]. However, it remains to be determined what other antagonisms may exist, by which different viruses antagonize individual SHREKs. Previous studies have shown that several of the SHREK proteins, such as PSGL-1 [5], CD43 [37], CD164 [34,38], TMEM123 [39], MUC1 [40], and MUC4 [41], are induced by interferons. However, SHREK proteins in general are likely different from interferon- induced restriction factors, and can be constitutively expressed in specific cell populations. Interestingly, we noticed that multiple SHREKs (CD34, PODXL1, PODXL2, and CD164) identified in this study are heavily expressed on the surface of stem and progenitor cells. In particular, CD34, the common surface marker of stem and progenitor cells, is one of the most potent SHREKs against retroviruses such as HIV-1 in our study (Figure2C). It is likely that these SHREKs are a part of host innate antiviral mechanisms that limit retroviral replication in critical cell populations such as stem and progenitor cells. It has been known that HIV-1 can enter and express in CD34+ multipotent hematopoietic progenitor Viruses 2021, 13, 832 13 of 15

cells (HPCs), but viral replication is limited [42]. However, viral replication can occur in HPCs with GM-CSF and TNF-α treatment, which induces myeloid differentiation. Such cytokine-induced viral permissiveness coincides with cytokine-mediated CD34 removal from the cell surface [42]. We found that several SHREKs (PSGL-1, CD164, TIM-1, MUC1, and MUC4) inhibited Ha-CoV-2 infection. MUC1 and MUC4 are closely related mucins; MUC4 had the strongest inhibition among these SHREKs in our infection assay. Purified cell-free human breast milk mucins have been shown to possess anti-HIV activity [43]. A recent study has suggested that MUC4 expression plays a protective role in female mice in SARS-CoV infection [44]. In addition, in chikungunya virus (CHIKV) infection, the loss of MUC4 also results in augmented disease during early time points, indicating a possible broad role for MUC4 in viral infection and pathogenesis [44]. We found no inhibition of Ha-CoV-2 by CD43, CD34, PODXL1, PODXL2, and TMEM123 (Supplementary Figure S13), although these proteins are potent inhibitors of HIV-1 (Figures2B and3). It is possible that the difference may result from the different sites of viral assembly and budding. SARS-CoV-2 budding occurs mainly at the membranes of ER-Golgi intermediate compartment [45], whereas HIV-1 buds from the plasma mem- brane [46]. Because of the differences, it is possible that different sets of cellular proteins may be incorporated into HIV-1 and SARS-CoV-2. Our identification of the SHREK family of proteins offers potential new antiviral therapeutics that may be developed through the induction and modulation of SHREK activities and the inhibition of viral antagonisms.

5. Patents A provisional patent application pertaining to the results presented in this paper has been filed by George Mason University.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/v13050832/s1, Figure S1: Effects of ICAM-1 and E-selectin on HIV-1 infectivity; Figure S2: Expression of SHREK proteins in HEK293T cells; Figure S3: Comparison of CD43 and PSGL-1 expres- sion levels in transfected HEK293T cells and Jurkat and CEM-SS T cells; Figure S4: Over-expression of PSGL-1 in CEM-SS T cells leads to inactivation of virion infectivity; Figure S5: Dose-dependent inhibition of HIV-1 by TIM-1; Figure S6: Dose-dependent inhibition of HIV-1 by CD34; Figure S7: Dose-dependent inhibition of HIV-1 by PODXL1; Figure S8: Dose-dependent inhibition of HIV-1 by CD164; Figure S9: Dose-dependent inhibition of HIV-1 by MUC1; Figure S10: Dose-dependent inhibi- tion of HIV-1 by MUC4; Figure S11: Dose-dependent inhibition of HIV-1 by TMEM123; Figure S12: Effects of TIM-1 on HIV-1 release: Figure S13: Lack of inhibition of Ha-CoV-2 virion infectivity by CD43, CD43, PODXL1, PODXL2, and TMEM123; Figure S14: Effects of SHREK proteins on Ha-CoV-2 virion release and S protein incorporation. Author Contributions: Experiments were designed by Y.W. Manuscript was written by Y.W. and edited by A.A. Experiments were performed by D.D., S.H., B.H., and L.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded in part by US Public Health Service grant 1R01AI148012 from NIAID to Y.W. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The published article includes all data generated or analyzed during this study. Acknowledgments: The authors wish to thank the NIH AIDS Reagent Program for reagents. Feng Li for providing influenza viral expression vectors, Jennifer Guernsey for editorial assistance. Conflicts of Interest: A provisional patent application pertaining to the results presented in this paper has been filed by George Mason University. Viruses 2021, 13, 832 14 of 15

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