MARCH8 Targets Cytoplasmic Lysine Residues of Various Viral Envelope Glycoproteins

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1. Version published to 10.1128/spectrum.00618-21

   Feb 23, 2022
2. 

   ScreenIT

   Apr 24, 2021

   SciScore for 10.1101/2021.04.20.440588: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were originally obtained from ATCC and routinely tested negative for mycoplasma contamination (PCR Mycoplasma Detection Set, Takara)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Fixed cells were incubated with anti-T7e epitope mouse monoclonal antibodies (Novagen, 69522-4) and anti-HA rabbit polyclonal antibodies (Sigma Aldrich, H6908).	anti-T7e suggested: None
   The secondary antibodies Alexa 488 donkey anti-mouse IgG (Molecular Probes, A-21202) and Alexa 568 donkey anti-rabbit IgG (Molecular Probes, A-10042) were used for …

   SciScore for 10.1101/2021.04.20.440588: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were originally obtained from ATCC and routinely tested negative for mycoplasma contamination (PCR Mycoplasma Detection Set, Takara)

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Fixed cells were incubated with anti-T7e epitope mouse monoclonal antibodies (Novagen, 69522-4) and anti-HA rabbit polyclonal antibodies (Sigma Aldrich, H6908).	anti-T7e suggested: None
   The secondary antibodies Alexa 488 donkey anti-mouse IgG (Molecular Probes, A-21202) and Alexa 568 donkey anti-rabbit IgG (Molecular Probes, A-10042) were used for double staining.	anti-mouse IgG suggested: (Molecular Probes Cat# A-21202, RRID:AB_141607)
   To detect intracellular RABV-P protein, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature and incubated with anti-RABV-P rabbit polyclonal antibody (Tobiume et al., 2009).	anti-RABV-P suggested: None
   The secondary antibodies Alexa 488 donkey anti-mouse IgG and Alexa 568 donkey anti-rabbit IgG were used for staining.	anti-rabbit IgG suggested: None
   To evaluate ubiquitination states, proteins immunoprecipitated with anti-ubiquitin mouse antibodies were subjected to Western blotting with anti-T7e rabbit antibodies.	anti-ubiquitin suggested: None
   Total cell lysate was also subjected to immunoblotting with anti-T7e rabbit antibodies and anti-HA rabbit polyclonal antibodies (Sigma Aldrich, H6908) to evaluate the expression levels of Env-T7es and HA-MARCH8.	anti-HA suggested: (Sigma-Aldrich Cat# H6908, RRID:AB_260070) HA-MARCH8 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell maintenance: 293T, HeLa, NIH3T3, HOS, and M8166+MARCH8 (Tada et al., 2015) cells were maintained under standard conditions.	293T suggested: None HeLa suggested: None
   To determine viral infectivity, 1 × 104 NIH3T3 cells or HeLa cells were incubated with 1 ng of p24 antigen of either arenavirus virus envelope (LCMV-GP)- or alphavirus E3-E2-6K-E1 envelope (derived from CHIKV or RRV)-pseudotyped HIV-1 luciferase reporter viruses, respectively.	NIH3T3 suggested: None
   Recombinant DNA
   Sentences	Resources
   DNA constructs: The envelope glycoprotein (Env)-deficient HiBiT-tagged HIV-1 proviral indicator construct pNL-Luc2-IN/HiBiT-E(-)Fin, the HIV-1 Gag-Pol expression plasmid pC-GagPol-RRE, the VSV-G expression plasmid pC-VSVg, the severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein expression plasmid pC-SARS-S, the SARS-CoV-2-S protein expression plasmid pC-SARS2-S, the HIV-1 Rev expression plasmid pCa-Rev, the MARCH8 expression plasmid pC-MARCH8, pC-HA-MARCH8, and the tyrosine motif-mutant pC-MARCH8-AxxL, the ACE2 expression plasmid pC-ACE2 and the TMPRSS2 expression plasmid pC-TMPRSS2, have previously been described elsewhere (Iwabu et al., 2009; Ozono et al., 2021; Ozono et al., 2020; Tada et al., 2015).	pC-GagPol-RRE suggested: None VSV-G suggested: RRID:Addgene_138479) pCa-Rev suggested: None pC-MARCH8 suggested: None pC-MARCH8-AxxL suggested: None
   The plasmid pC-LCMVgp expressing the viral envelope derived from lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) was created by inserting PCR-amplified and BsiWI/XhoI-digested LCMV-GP fragments (PCR-amplified from pCI-LCMV-GP (Reignier et al., 2006)) into the Acc65I/XhoI-digested pCAGGS mammalian expression plasmid.	pCI-LCMV-GP suggested: None pCAGGS suggested: RRID:Addgene_18926)
   The plasmid pC-CHIKVe expressing alphavirus E3-E2-6K-E1 polyprotein derived from Chikungunya virus (CHIKV) was created by inserting the PCR-amplified and Acc65I/XhoI-digested E3-E2-6K-E1 fragments (PCR-amplified from codon-optimized CHIKV’s C-E3-E2-6K-E1 plasmid (Kishishita et al., 2013)) into the corresponding site of pCAGGS.	pC-CHIKVe suggested: None C-E3-E2-6K-E1 suggested: None
   Another E3-E2-6K-E1 expression plasmid, pC-RRVe, derived from the Ross River virus (RRV) T48 strain, was created by inserting the PCR-amplified and EcoRV/NotI-digested E3-E2-6K-E1 fragments (PCR-amplified from pRRV-E2E1 (Sharkey et al., 2001)) into the corresponding site of pCAGGS.	pRRV-E2E1 suggested: None
   The RABV-G or LCMV-GP mutant (pC-RABVg-CT3K/R, or pC-LCMVgp-CT6K/R), in which cytoplasmic lysine residues at positions 489/508/517 or 465/471/478/487/492/496 were mutated to arginine residues, was created by inserting overlapping PCR fragments into correspondingly digested pC-RABVg or pC-LCMVgp, respectively	pC-RABVg-CT3K/R suggested: None pC-LCMVgp-CT6K/R suggested: None
   Mutants of SARS-CoV-S and SARS-CoV-2-S (pC-SARS-S-CT4K/R, or pC-SARS2-S-CT4K/R), in which cytoplasmic lysine residues at positions 1227/1237/1248/1251 and 1245/1255/1266/1269 were mutated to arginine residues, were created by inserting overlapping PCR fragments into correspondingly digested pC-SARS-S and pC-SARS2-S, respectively.	pC-SARS-S-CT4K/R suggested: None pC-SARS2-S-CT4K/R suggested: None pC-SARS-S suggested: None
   The CHIKV and RRV 6K mutants (pC-CHIKV-6K-2K/R and pC-RRV-6K-2K/R), in which the 6K region’s cytoplasmic lysine residues at positions 37/44 were mutated to arginine residues, were created by inserting overlapping PCR fragments into correspondingly digested pC-CHIKVe and pC-RRVe, respectively.	pC-CHIKV-6K-2K/R suggested: None pC-RRV-6K-2K/R suggested: None pC-RRVe suggested: None
   Similarly, the E2 mutants of CHIKV and RRV (pC-CHIKVe-E2-K/R or pC-RRVe-E2-K/R), in which E2’s C-terminal lysine residues at positions 422 and 394 were mutated to arginine residues, were created by inserting overlapping PCR fragments into correspondingly digested pCAGGS.	pC-CHIKVe-E2-K/R suggested: None pC-RRVe-E2-K/R suggested: None
   The C-terminally T7-epitope–tagged wild-type and its mutant expression plasmids of RABV-G (pC-RABVg-T7e and pC-RABVg-CT3K/R-T7e), LCMV-GP (pC-LCMVgp-T7e and pC-LCMVgp-CT6K/R-T7e), SARS-CoV-S (pC-SARS-S-T7e and pC-SARS-S-CT4K/R-T7e), SARS-CoV-2-S (pC-SARS2-S-T7e and pC-SARS2-S-CT4K/R-T7e), CHIKVe3-E2-6K-E1 (pC-CHIKVe-T7e, pC-CHIKVe-6K-2K/R-T7e, and pC-CHIKVe-E2-K/R-T7e), and RRVe3-E2-6K-E1 (pC-RRVe-T7e, pC-RRVe-6K-2K/R-T7e, and pC-RRVe-E2-K/R-T7e) were created by replacing the VSV-G gene of pC-VSVg-T7e (Zhang et al., 2020) with the corresponding PCR-amplified fragments.	pC-RABVg-T7e suggested: None pC-LCMVgp-T7e suggested: None pC-LCMVgp-CT6K/R-T7e suggested: None pC-SARS-S-T7e suggested: None pC-SARS-S-CT4K/R-T7e suggested: None pC-SARS2-S-T7e suggested: None pC-SARS2-S-CT4K/R-T7e suggested: None pC-CHIKVe-T7e suggested: None pC-CHIKVe-6K-2K/R-T7e suggested: None pC-CHIKVe-E2-K/R-T7e suggested: None pC-RRVe-T7e suggested: None pC-RRVe-6K-2K/R-T7e suggested: None pC-RRVe-E2-K/R-T7e suggested: None pC-VSVg-T7e suggested: None
   To detect ubiquitinated CHIKV-E2 proteins by immunoprecipitation assays, WT or E2-K/R mutant plasmids expressing CHIKV E3-E2-6K-E1, in which the T7 epitope-tag is inserted immediately downstream of a furin-cleavage site between E3 and E2, were created by inserting overlapping PCR fragments that harbor a T7e sequence into correspondingly digested pCAGGS and were designated pC-CHIKVe-T7eE2, pC-CHIKVe-T7eE2-K/R, pC-RRVe-T7eE2, and pC-RRVe-T7eE2-K/R, respectively.	pC-RRVe-T7eE2 suggested: None pC-RRVe-T7eE2-K/R suggested: None
   Virion infectivity assays: To prepare various viral envelope-pseudotyped HIV-1 luciferase reporter viruses, 1.1 × 105 293T cells were cotransfected with increasing amounts of the MARCH8 expression plasmid, 20 ng of viral envelope expression plasmid (pC-VSVg, pC-RABVg, pC-LCMVgp, pC-SARS-S, pC-SARS2-S, pC-CHIKVe, pC-RRVe, and their mutants), 500 ng of pNL-Luc2-IN/HiBiT-E(-)Fin, and an empty vector up to 1 μg of total DNA using FuGENE 6 (Promega).	pC-VSVg suggested: None pC-RABVg suggested: None pC-LCMVgp suggested: None pC-SARS2-S suggested: None pNL-Luc2-IN/HiBiT-E(-)Fin suggested: None
   Alternatively, 2.2 × 104 293T cells transiently coexpressing ACE2 and TMPRSS2 (using pC-ACE2 and pC-TMPRSS2) were incubated with 1 ng of p24 antigen of either SARS-CoV-S- or SARS-CoV-2-S-pseudotyped luciferase reporter lentiviruses.	pC-ACE2 suggested: None pC-TMPRSS2 suggested: None
   Immunofluorescence microscopy: HOS cells were plated on 13-mm coverslips, cotransfected with 0.5 μg of pC-xx-T7e (xx: RABV-G, RABV-G-CT3K/R, LCMVgp, LCMVgp-CT6K/R, SARS-S, SARS-S-CT4K/R, SARS2-S, SARS2-S-CT4K/R, CHIKVe, CHIKVe-6K-2K/R, CHIKVe-E2-K/R, RRVe, RRVe-6K-2K/R, or RRVe-E2-K/R), 0.1 μg of pC-GagPol-RRE, 0.05 μg of pCa-Rev, and 0.3 μg of either the HA-MARCH8 expression plasmid or an empty control using FuGENE6, and cultured for 24 h.	pC-xx-T7e suggested: None HA-MARCH8 suggested: None
   Ubiquitination assays: 293T cells (5 × 105) were cotransfected with 0.8 μg of pC-RABVg-T7e, pC-RABVg-CT3K/R-T7e, pC-CHIKVe-T7eE2, or pC-CHIKVe-T7eE2-K/R; and 0.2 μg of pC-HA-MARCH8 or an empty control.	pC-RABVg-CT3K/R-T7e suggested: None pC-CHIKVe-T7eE2 suggested: None pC-CHIKVe-T7eE2-K/R suggested: None pC-HA-MARCH8 suggested: None
   Software and Algorithms
   Sentences	Resources
   Statistical analyses: Column graphs that combine bars and individual data points were created with GraphPad Prism version 9.10.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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3. 

   Version published to 10.1101/2021.04.20.440588v1 on bioRxiv

   Apr 21, 2021