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

Abstract

<jats:title>Abstract</jats:title><jats:p>HIV-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.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>The HIV-1 Env cytoplasmic tail (CT) is required for efficient Env incorporation into nascent particles and viral transmission in primary CD4<jats:sup>+</jats:sup> 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.</jats:p></jats:sec>

Rab11-FIP1C is dispensable for HIV-1 replication in primary CD4+T cells but its role is cell-type-dependent in immortalized human T-cell lines

“…FIP1C expression in HeLa and 293T was approximately equivalent, while FIP1C protein levels in the T-cell lines tested – SupT1, C8166, and M8166 – varied. Consistent with a previous report (65), and despite being highly permissive for HIV-1 replication (64, 66), MT-4 cells did not express detectable levels of FIP1C. All four T-cell lines expressed Rab14.…”

“…To examine whether FIP1C and Rab14 expression may be influenced by HTLV-I transformation and determine their expression levels across previously un-evaluated T-cell lines permissive to HIV-1 replication, FIP1C and Rab14 expression in other HTLV-1-transformed T-cell lines was evaluated (Figure 2C). Western blotting was performed to measure FIP1C and Rab14 expression in several adult T-cell leukemia/lymphoma (ATL) cells that express little or no detectable Tax protein (Tax − ) (64), though some of these cell lines (e.g., TL-Om1 and ED) were permissive to HIV-1 replication (69). The FIP1C-deficient MT-4 and FIP1C-expressing SupT1 were included as negative and positive controls, respectively.…”

“…HIV-1 replication in most T-cell lines is abrogated by truncation of the gp41 CT (37, 38, 63, 64). Several studies have shown that mutating a hydrophobic motif, Tyr 795 Trp (Y 795 W), located in the LLP3 domain of the gp41 CT, to Ser-Leu (Y 795 W/SL; Figure.…”

“…H9 cells were obtained from Robert Gallo (Cat# ARP-87) through the NIH-ARP and STR profiled to confirm their identity and was a 100% match to the Cellosaurus cell line accession ID CVCL_1240. The SupT1-CCR5 (76) (referred to as SupT1huR5; cell line accession ID on the Cellosaurus database is CVCL_X633) T-cell line was a generous gift from James Hoxie [cell line accession ID on the Cellosaurus database is CVCL_X633; STR profile published in (64)]. The ATL Tax − cells lines (ED, ATL-55T, and TL-Om1) were a generous gift from Chou-Zen Giam.…”

“…Virus replication kinetics were determined as previously described (64). Briefly, 2.5×10 6 T cells were transfected with proviral clones (1 μg DNA/10 6 cells) in the presence of 700 μg/mL DEAE-dextran and cultured in 3 mL of RPMI-10% FBS.…”