Eparticles to the plasma membrane (PM), including retroviruses, Ebola, Kaposi sarcoma-associated herpes virus (KSHV) and influenza virus like particles [9,26?8]. Vpu-mediated antagonism of tetherin requires an interaction between the MSDs of Vpu and tetherin, but as of yet, there is no consensus on the precise mechanism by which Vpu modulates tetherin activity. Vpu has been reported to reduce tetherin surface expression by altering the rate of recycled and/or restricting newly synthesized tetherin from reaching the PM [29?4]. However, it has also been reported that Vpu can modulate tetherin AZP-531 site activity in the absence of surface downmodulation and intracellular depletion [35]. Some studies suggest that tetherin can be degraded through b-TrCP mediated targeting to lysosomes or the proteasome [33,36,37]. Although the mechanisms for CD4 and tetherin antagonism are believed to be distinct, evidence suggests that Vpu contains some shared features required for modulation of both proteins. For instance, complete proscription of either target requires two critical serines housed in the Vpu cytoplasmic tail, which is also required for interaction with b-TrCP and degradation of tetherinVpu Modulation of Distinct Targetsor CD4 [17,37,38]. Vpu AZP-531 mutants lacking these serine residues retain some activity against tetherin but not CD4 [34,39]. Direct parallels between Vpu modulation of tetherin and CD4 are difficult to draw due to differences and limitations in the assays employed. Studies investigating tetherin antagonism have relied heavily on detection of viral particle release, through protein release or infectious virus production, although some studies have also measured tetherin modulation directly. Reports on CD4 11138725 down-modulation typically rely on biochemical assays measuring total protein or surface expression. Additionally, Vpu studies have used different cell types, multiple methods of introducing CD4 or tetherin targets (endogenous or exogenous), and different methods of producing Vpu (e.g., native or codon-optimized, contained in the provirus or introduced in trans). Employment of these disparate protocols limits the ability to directly compare different studies. We and others found that Vpu prevents GaLV Env incorporation into HIV-1 particles, likely through a shared structural recognition motif INxxIxxVKxxVxRxK in the Env cytoplasmic tail that resembles the critical Vpu sensitivity motif found in the cytoplasmic tail of CD4 [1?]. This motif is conserved and is transferrable to confer sensitivity in previously insensitive proteins [1]. Based on these findings, we currently believe Vpu mistakenly recognizes the cytoplasmic tail of GaLV Env as a CD4 analogue. Similarly, GaLV Env is packaged into the virus in the absence of Vpu, however, unlike CD4, GaLV Env can form infectious pseudotyped virus to assess incorporation of the target protein. Modulation of GaLV Env by Vpu is sensitive and well suited for a comparative study with the modulation of tetherin by Vpu. By employing GaLV Env, constraint of both distinct targets can be studied in the same cell type using Vpu encoded in the provirus with infectivity as the output for both.Infectivity analysis293FT cells were plated in 6-well plates and allowed to reach 60 confluency prior to transfection. For tetherin studies, 293FT cells were 1662274 transfected with the following expression constructs: provirus (425 ng) and VSV-G (25 ng) with or without 12.5 ng of tetherin-HA in a total of 500 ng. For GaLV Env a.Eparticles to the plasma membrane (PM), including retroviruses, Ebola, Kaposi sarcoma-associated herpes virus (KSHV) and influenza virus like particles [9,26?8]. Vpu-mediated antagonism of tetherin requires an interaction between the MSDs of Vpu and tetherin, but as of yet, there is no consensus on the precise mechanism by which Vpu modulates tetherin activity. Vpu has been reported to reduce tetherin surface expression by altering the rate of recycled and/or restricting newly synthesized tetherin from reaching the PM [29?4]. However, it has also been reported that Vpu can modulate tetherin activity in the absence of surface downmodulation and intracellular depletion [35]. Some studies suggest that tetherin can be degraded through b-TrCP mediated targeting to lysosomes or the proteasome [33,36,37]. Although the mechanisms for CD4 and tetherin antagonism are believed to be distinct, evidence suggests that Vpu contains some shared features required for modulation of both proteins. For instance, complete proscription of either target requires two critical serines housed in the Vpu cytoplasmic tail, which is also required for interaction with b-TrCP and degradation of tetherinVpu Modulation of Distinct Targetsor CD4 [17,37,38]. Vpu mutants lacking these serine residues retain some activity against tetherin but not CD4 [34,39]. Direct parallels between Vpu modulation of tetherin and CD4 are difficult to draw due to differences and limitations in the assays employed. Studies investigating tetherin antagonism have relied heavily on detection of viral particle release, through protein release or infectious virus production, although some studies have also measured tetherin modulation directly. Reports on CD4 11138725 down-modulation typically rely on biochemical assays measuring total protein or surface expression. Additionally, Vpu studies have used different cell types, multiple methods of introducing CD4 or tetherin targets (endogenous or exogenous), and different methods of producing Vpu (e.g., native or codon-optimized, contained in the provirus or introduced in trans). Employment of these disparate protocols limits the ability to directly compare different studies. We and others found that Vpu prevents GaLV Env incorporation into HIV-1 particles, likely through a shared structural recognition motif INxxIxxVKxxVxRxK in the Env cytoplasmic tail that resembles the critical Vpu sensitivity motif found in the cytoplasmic tail of CD4 [1?]. This motif is conserved and is transferrable to confer sensitivity in previously insensitive proteins [1]. Based on these findings, we currently believe Vpu mistakenly recognizes the cytoplasmic tail of GaLV Env as a CD4 analogue. Similarly, GaLV Env is packaged into the virus in the absence of Vpu, however, unlike CD4, GaLV Env can form infectious pseudotyped virus to assess incorporation of the target protein. Modulation of GaLV Env by Vpu is sensitive and well suited for a comparative study with the modulation of tetherin by Vpu. By employing GaLV Env, constraint of both distinct targets can be studied in the same cell type using Vpu encoded in the provirus with infectivity as the output for both.Infectivity analysis293FT cells were plated in 6-well plates and allowed to reach 60 confluency prior to transfection. For tetherin studies, 293FT cells were 1662274 transfected with the following expression constructs: provirus (425 ng) and VSV-G (25 ng) with or without 12.5 ng of tetherin-HA in a total of 500 ng. For GaLV Env a.