Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: CD4 incorporation into virions. ( A ). Media samples from 293 cells transfected with psPAX2 (psPAX) plus the CD4 or CD4-gpi expression constructs were pelleted through 20% sucrose cushions, separated by electrophoresis and immunoblotted for detection of CA and CD4 immunoreactive proteins. PrGag, p41 (Gag intermediate), CA, and CD4 proteins were identified based on their immunoreactivities and migration mobilities relative to protein molecular weight standards run in parallel. ( B , C ). Cell samples (top panels) and media samples that were pelleted through 30% sucrose cushions (bottom panels) were collected from cells transfected with the indicated constructs, separated by electrophoresis, and immunoblotted for detection of PrGag, p41 (Gag intermediate) and CA ( B ) or CD4 and CD4-gpi ( C ). Note that viral CD4-to-CA ratios (expressed as percentages) were 33% for ( A ), and 25% for ( B , C ); while viral CD4-gpi-to-CA ratios were 26% for ( A ), and 34% for ( B , C ).

Article Snippet: The lentivirus GagPol packaging plasmid (psPAX2), the GFP expression plasmids (GFP-PS, GFP-NoPS, GFP-C1-PLCdelta-PH, pEGFP-GFP11-ClathrinLightChain, pcDNA3.1-GFP [ , , , , , , , , , ]), the VSV G expression vector pMD.G, the CD4 expression plasmid pCAGGS-CD4-Myc, and the CXCR4 expression plasmid pcDNA3-CXCR4 were all obtained from Addgene, and were the gifts of Drs. Didier Trono, Jennifer Doudna, Tobias Meyer, Bo Huang, Simon Davis, Erik Procko, and Jacob Yount [ , , , , , , ].

Techniques: Transfection, Expressing, Construct, Electrophoresis, Migration, Molecular Weight

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Infection of HIV-1-infected cells. Jurkat T cells, HIV-1-infected J1.1 Jurkat cells, and J1.1 cells induced with tumor necrosis factor alpha (J1.1 + TNFa) were subjected to immunoblot detection of Gag proteins ( A ) with an anti-CA antibody, immunoblot detection of Env proteins ( B ) with an anti-gp41(TM) antibody, and infection with a lentivirus vector ( C ) generated by transfection of 293 cells with psPAX2, pLVX-puro-Xho-ATG-β-gal, CD4-GPI, and CXCR4-328 plasmids. ( A , B ) Migration positions of PrGag, CA, gp160 and gp41 proteins are indicated, as are molecular weight standards (in kDa) that were electrophoresced in parallel. ( C ) Infectivity results represent normalized transduced β-gal activity readings normalized to results for induced J1.1 cells. Averages and standard deviations derive from duplicate (Jurkat, J1.1) or triplicate (J1.1 + TNFα) infections, and differences between Jurkat and J1.1, as well as J1.1 versus J1.1 + TNFα were highly significant ( p < 0.001), as determined by conversion of means and standard deviations to Z values that were used in probability calculations.

Article Snippet: The lentivirus GagPol packaging plasmid (psPAX2), the GFP expression plasmids (GFP-PS, GFP-NoPS, GFP-C1-PLCdelta-PH, pEGFP-GFP11-ClathrinLightChain, pcDNA3.1-GFP [ , , , , , , , , , ]), the VSV G expression vector pMD.G, the CD4 expression plasmid pCAGGS-CD4-Myc, and the CXCR4 expression plasmid pcDNA3-CXCR4 were all obtained from Addgene, and were the gifts of Drs. Didier Trono, Jennifer Doudna, Tobias Meyer, Bo Huang, Simon Davis, Erik Procko, and Jacob Yount [ , , , , , , ].

Techniques: Infection, Western Blot, Plasmid Preparation, Generated, Transfection, Migration, Molecular Weight, Activity Assay

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Effects of lipid variations on fusion in reverse infections. ( A ) The HIV-1 viral membrane is highly enriched in cholesterol (CHOL), and has substantial amounts of ceramide (CER). The inner leaflets are enriched for phosphatidylserine (PS), phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2; PIP2), and phosphatidylethanolamine (PE), while the outer leaflet is enriched in sphingomyelin (SM), hexosylceramides (HEX), and saturated phosphatidylcholines (PCs). ( B – D ) Infectivities of equivalent volumes of virus were scored via β-gal activities, and were normalized either to untreated (−) samples, or for convenience in the panel ( D ) CD/CX infections, to viruses from CerS−/− cells. In all panels, CD/CX viruses refer to lentivirus vectors generated by co-transfection of 293 cells with a GagPol expression vector (psPAX2), a β-gal-transducing vector (pLVX-puro-Xho-ATG-β-gal), as well as CD4-GPI and CXCR4-318 plasmids. Also, in all panels, Env-WT and Env-753 targets cells refer to 293 cells transfected to express those proteins. For panels ( B , C ), WT and 753 viruses are the NL4-3 variants and were used to infect TZM-bl reporter cells; for panel ( D ), WT refers to viruses produced from cells transfected with psPAX2, pLVX-puro-Xho-ATG-β-gal, plus a WT Env expression vector, while targets were 293 cells transfected to express the CD4-GPI and CXCR4-318 proteins. In panel ( B ), AME refers to treatment of viruses with 10 mM AME prior to infections. In panel ( C ), TMEM refers to co-transfection of virus producing cells with (+) or without (−) the mTMEM-GY scramblase expression vector. In panel ( D ), CerS2−/− pertains to producing viruses either from 293 cells (−) or from 293-CerS2−/− knockout cells (+). For panels ( B – D ), averages and standard deviations are as indicated, noting that where error bars are missing, it is because the standard deviations were too small to be depicted. The following significant differences were obtained by conversion of means and standard deviations to Z values for probability calculations: Panel ( B ) WT/TZMBL, AME −/+, p < 0.001; Panel ( B ) CDCX/Env-WT, AME −/+, p < 0.001; Panel C all pairs p < 0.001; Panel ( D ) CDCX/Env-WT, CerS2−/−, −/+, p < 0.01; all other Panel D pairs, p < 0.001. Note that in panel ( D ), if normalizations were based on CDCX viruses produced from WT 293 cells and infections of Env-753Stop-expressing cells, infectivities of CDCX viruses from virus–cell combinations would be as follows: from WT 293 cells into Env-753Stop cells, 100%; from CerS2−/− cells into Env-753Stop cells 500%; from WT 293 cells into Env-WT cells, 8%; from CerS2−/− cells into Env-WT cells, 19%.

Article Snippet: The lentivirus GagPol packaging plasmid (psPAX2), the GFP expression plasmids (GFP-PS, GFP-NoPS, GFP-C1-PLCdelta-PH, pEGFP-GFP11-ClathrinLightChain, pcDNA3.1-GFP [ , , , , , , , , , ]), the VSV G expression vector pMD.G, the CD4 expression plasmid pCAGGS-CD4-Myc, and the CXCR4 expression plasmid pcDNA3-CXCR4 were all obtained from Addgene, and were the gifts of Drs. Didier Trono, Jennifer Doudna, Tobias Meyer, Bo Huang, Simon Davis, Erik Procko, and Jacob Yount [ , , , , , , ].

Techniques: Membrane, Virus, Generated, Cotransfection, Expressing, Plasmid Preparation, Transfection, Produced, Knock-Out