Journal: mBio

Article Title: AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4

doi: 10.1128/mbio.03382-22

Figure Lengend Snippet: Nef strongly enhances HIV-1 replication in MOLT-3 cells lacking LCK. (A) LCK expression in parental MOLT-3 cells and in LCK KO clones analyzed by Western blotting. (B) CD4 surface levels on the same cells analyzed by flow cytometry. (C) Western blots showing the effects of Nef on HIV-1 replication in parental MOLT-3 cells and in LCK KO clones. The cells were infected with equal amounts (0.2 ng p24/mL) of Nef + or Nef − HIV-1 NL4-3 , and cell lysates were examined with anti-CA and anti-actin 10 days after infection. (D) Virus replication in the same cultures monitored by measuring p24 accumulation in the supernatants over time.

Article Snippet: MOLT-3 cells were obtained from the ATCC.

Techniques: Expressing, Clone Assay, Western Blot, Flow Cytometry, Infection

Journal: mBio

Article Title: AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4

doi: 10.1128/mbio.03382-22

Figure Lengend Snippet: Nef can strongly enhance HIV-1 replication in cells lacking PAK2. (A) PAK2 expression in parental and in PAK2 KO MOLT-3 cells analyzed by Western blotting. (B) CD4 surface levels on the same cells analyzed by flow cytometry. (C) Western blots showing that the effects of Nef on HIV-1 replication in parental and in PAK2 KO MOLT-3 cells are comparable. The cells were infected with 0.2 ng p24/mL of Nef + or Nef − HIV-1 NL4-3 , and cell lysates were analyzed as in Fig. 2C . (D) Virus replication monitored in parallel by measuring p24 accumulation in the supernatants.

Article Snippet: MOLT-3 cells were obtained from the ATCC.

Techniques: Expressing, Western Blot, Flow Cytometry, Infection

Journal: mBio

Article Title: AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4

doi: 10.1128/mbio.03382-22

Figure Lengend Snippet: Nef can fully support HIV-1 replication in the absence of all group I PAKs. (A) Expression of group I PAK mRNAs in MOLT-3 cells quantified by transcriptome sequencing (RNA-seq) as fragments per kilobase of transcript per million mapped reads (FPKM). (B) Expression of PAK1 and PAK2 in parental MOLT-3 cells and in PAK1/PAK2 double-KO clones analyzed by Western blotting. (C) CD4 surface levels on the same cells analyzed by flow cytometry. (D and E) Replication of Nef + and Nef − HIV-1 NL4-3 in the same cells after infection with 0.2 ng/mL p24, monitored by Western blotting of cell lysates with anti-CA (D) and by measuring p24 accumulation in the supernatants (E).

Article Snippet: MOLT-3 cells were obtained from the ATCC.

Techniques: Expressing, Sequencing, RNA Sequencing Assay, Clone Assay, Western Blot, Flow Cytometry, Infection

Journal: mBio

Article Title: AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4

doi: 10.1128/mbio.03382-22

Figure Lengend Snippet: Nef remains required for efficient HIV-1 replication in MOLT-3 cells lacking SERINCs and Nef-sensitive CD4. (A) CD4 surface levels on parental MOLT-3 cells and on M3 triple-KO (SERINC3/SERINC5/CD4 KO) cells analyzed by flow cytometry. (B) CD4 surface levels on M3-triple-KO/CD4 and M3 triple-KO/CD4 ΔCT cells stably transduced with empty MSCVpuro (vector) or with a version expressing Nef LAI . (C) Relative infectivities of Nef + and Nef − HIV-1 NL4-3 virions produced in M3 triple-KO/CD4 ΔCT cells measured using MOLT-3/ZsGreen reporter cells. Data are mean of three experiments with SD. (D) Replication of Nef + and Nef − HIV-1 NL4-3 in M3 triple-KO/CD4 ΔCT cells. The cells were infected with 0.2 ng p24/mL, and cell lysates were examined by Western blotting with anti-CA and anti-actin on day 8 postinfection (p.i.). (E) Replication of Nef + and Nef − versions of the R5-tropic NL-JRFL and NL-ZM109 viruses in M3 triple-KO/CD4 ΔCT /CCR5 cells infected with 0.2 ng p24/mL, monitored by measuring p24 accumulation in the supernatants. (F) Replication of Nef + and Nef − HIV-1 NL4-3 in heterokaryons formed between M3 triple-KO/CD4 ΔCT (GFP 8-11 ) cells and JTAg double-KO(GFP 1-7 ) cells transiently expressing the HN and F proteins of NDV. GFP-positive cells were sorted by FACS and infected with 5 ng p24/mL. Virus replication was monitored by measuring p24 accumulation in the supernatants.

Article Snippet: MOLT-3 cells were obtained from the ATCC.

Techniques: Flow Cytometry, Stable Transfection, Transduction, Plasmid Preparation, Expressing, Produced, Infection, Western Blot

Journal: mBio

Article Title: AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4

doi: 10.1128/mbio.03382-22

Figure Lengend Snippet: Replication of Nef mutants in M3 triple-KO/CD4 ΔCT cells lacking SERINCs and Nef-sensitive CD4. (A) Effect of a mutation (WL57,58AA) reported to disrupt binding of Nef to the cytoplasmic domain of CD4. (B) Effects of mutations that disrupt AP-2 binding. (C) Effects of mutations in a conserved diacidic motif that is specifically required for binding to AP-2 but not AP-1 or AP-3. Of note, AP-2 binding is unaffected by the conservative D174E substitution but is impaired by the equally conservative D175E substitution . (D) Side-by-side comparison of the abilities of the Nef mutants to spread in parental MOLT-3 cells and in M3 triple-KO/CD4 ΔCT cells. The cells were infected with WT (Nef + ) HIV-1 NL4-3 or with the indicated Nef mutants (0.2 ng p24/mL), and virus replication was monitored by measuring p24 accumulation in the supernatants (A to C) or by Western blotting of cell lysates with anti-CA (D). The virus replication curves are all from the same experiment.

Article Snippet: MOLT-3 cells were obtained from the ATCC.

Techniques: Mutagenesis, Binding Assay, Infection, Western Blot