Replication of HIV-1 envelope protein cytoplasmic domain variants in permissive and restrictive cells

Staubus, August O.; Alfadhli, Ayna; Barklis, Robin Lid; Barklis, Eric

doi:10.1016/j.virol.2019.09.008

Cited by 10 publications

(15 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Importantly, while CerS2−/− cells supported the efficient assembly and release of viruses containing normal amounts of HIV-1 Env proteins, viruses so obtained were a third as infectious as those produced in WT cells. Similar results were obtained with HIV-1 virions carrying an Env protein cytoplasmic domain deletion (ΔCT; ( 17 , 25 , 26 )), but infectivities of HIV-1 virions pseudotyped ( 27 , 28 ) with the vesicular stomatitis virus (VSV) glycoprotein (G) were not so affected. Cell–cell fusion assay results mimicked virus infection results, indicating that the Env protein defect in CerS2−/− cells was independent of other virally encoded constituents.…”

supporting

confidence: 57%

“…4 B ). We also tested two additional HIV-1 Env CT mutants: one of these was an Env P714L mutant (also called P203L ( 14 , 15 )) that was selected for resistance to cholesterol depletion; and the other was the Env 716Ins-R∗ mutant, which is cleaved by the HIV-1 protease (PR) in a similar fashion to P714L ( 26 ). These variants also showed significant infectivity reductions for viruses produced in mutant versus WT HEK293T cells (CerS2−/− P714L inf/Gag = 6% WT cells; CerS2−/− 716Ins-R∗ = 30 ± 11% WT cells).…”

Section: Resultsmentioning

confidence: 99%

“…WT HIV-1 virus stocks were generated by transfection of 10 cm plates of confluent cells with 24 μg of the HIV-1 proviral NL4-3 plasmid ( 33 ) using polyethyleneimine ( 26 , 42 ), and collecting cell lysates and 0.45 micron-filtered viral supernatants at 72 h post-transfection as described previously ( 26 , 36 , 41 , 42 , 62 ). Mutant HIV-1 stocks similarly were prepared by transfection with NL4-3–derived Env ΔCT (CT144 ( 25 , 26 ); obtained from Dr Eric Freed, NCI Frederick), Env P714L (P203L ( 14 , 15 ), obtained from Dr Eric Freed, NCI Frederick), and Env 716Ins-R∗ ( 26 ) constructs. For pseudotyping of HIV-1 viruses with the VSV G, 10 cm plates of cells were transfected with 16 μg of HIV-Luc, an env -deleted HIV-1 expression plasmid ( 40 , 41 ), plus 8 μg of the VSV G expression plasmid pMD.G (Addgene plasmid #12259; kindly provided by Dr Didier Trono).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Ceramide synthase 2 deletion decreases the infectivity of HIV-1

Barklis

Alfadhli

Kyle

et al. 2021

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell–cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.

show abstract

supporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Ceramide synthase 2 deletion decreases the infectivity of HIV-1

Barklis

Alfadhli

Kyle

et al. 2021

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…1) and previously reported permissivity, or lack thereof, to gp41 CT truncation. Previous studies have reported that MT-4 (14,21,29,31,33,38,(41)(42)(43)(44)(45) and M8166 (58) are permissive to truncation of the gp41 CT, and it is established that hPBMCs, SupT1, and Jurkat E6.1 are not permissive (14,21). Therefore, two HTLV-1lymphoma-derived cell lines, SupT1 (59) and Jurkat E6.1 (60) (Fig.…”

Section: Lineage and Validation Of Cell Lines Used To Interrogate T-cmentioning

confidence: 97%

“…HIV-1 replication in most T-cell lines is abrogated by truncation of the gp41 CT (14,21), but several studies have demonstrated that the MT-4 cell line is able to propagate a gp41 CT-deleted mutant (14,21,29,31,33,38,(41)(42)(43)(44)(45). To evaluate the ability of the CTdel144 mutant, which lacks 144 amino acids from the gp41 CT (26), to replicate in HTLV-I-transformed T-cell lines, MT-4, C8166 and M8166 cells were transfected with the WT pNL4-3 molecular clone or the CTdel144 derivative, to initiate a spreading infection (Fig.…”

Section: Mt-4 Is the Only T-cell Line Tested In This Study That Is Pementioning

confidence: 99%

Elucidating the basis for permissivity of the MT-4 T-cell line to replication of an HIV-1 mutant lacking the gp41 cytoplasmic tail

Fernandez

Hoffman

Pezeshkian

et al. 2020

Preprint

View full text Add to dashboard Cite

AbstractHIV-1 encodes an envelope glycoprotein (Env) that contains a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virus particles and viral transmission. In most physiologically relevant cell types, the gp41 CT is required for HIV-1 replication, but in the MT-4 T-cell line the gp41 CT is not required for a spreading infection. To help elucidate the role of the gp41 CT in HIV-1 transmission, in this study we investigated the viral and cellular factors that contribute to the permissivity of MT-4 to gp41 CT truncation. We found that the kinetics of HIV-1 production are faster in MT-4 than in the other T-cell lines tested, but MT-4 express equivalent amounts of HIV-1 proteins on a per-cell basis relative to cells not permissive to CT truncation. MT-4 express higher levels of plasma-membrane-associated Env than non-permissive cells and Env internalization from the plasma membrane is slower compared to another T-cell line, SupT1. Paradoxically, despite the high levels of Env on the surface of MT-4, two-fold less Env is incorporated into virus particles in MT-4 compared to SupT1. Cell-to-cell transmission between co-cultured 293T and MT-4 is higher than in co-cultures of 293T with most other T-cell lines tested, indicating that MT-4 are highly susceptible to this mode of infection. These data help to clarify the long-standing question of how MT-4 cells overcome the requirement for the HIV-1 gp41 CT and support a role for gp41 CT-dependent trafficking in Env incorporation and cell-to-cell transmission in physiologically relevant cell lines.ImportanceThe HIV-1 Env cytoplasmic tail (CT) is required for efficient Env incorporation into nascent particles and viral transmission in primary CD4+ T cells. The MT-4 T-cell line has been reported to support multiple rounds of infection of HIV-1 encoding a gp41 CT truncation. Uncovering the underlying mechanism of MT-4 T-cell line permissivity to gp41 CT truncation would provide key insights into the role of the gp41 CT in HIV-1 transmission. This study reveals that multiple factors contribute to the unique ability of a gp41 CT truncation mutant to spread in cultures of MT-4 cells. The lack of a requirement for the gp41 CT in MT-4 is associated with the combined effects of rapid HIV-1 protein production, high levels of cell-surface Env expression, and increased susceptibility to cell-to-cell transmission compared to non-permissive cells.

show abstract