Ckbones infectious molecular clones (Env-IMC), refered here as “NL-Env.ecto”. To avoid creating chimerism in any reading frames overlapping env, heterologous env sequences encoding the ectodomain were cloned into the NL4-3 backbone, wherein the recombinant viruses express full-length Env [4]. We used three R5-tropic reference HIV-1 MedChemExpress 16960-16-0 variant (NL-SF162.ecto, NL-YU-2.ecto, and NL-BaL.ecto), as well as eight viruses encoding CCR5-utilizing env genes from clade B mucosally transmitted T/F HIV-1 variants, including NL-CH077.ecto, NL-1051.TD12.ecto, NL-1051.C22.ecto, NL-TT31P.2G1.ecto, NL-TT31P.2F10.ecto, NL-SC22.3C2.ecto, NL-RHPA.ecto, and NL-9010.A1.ecto, with env genes derived, respectively, from subjects 700010077 (male; single variant transmission); 1051-12 (female; infected with two related variants); TT31P (female; infected with two related variants); SC22 (female; single variant transmission), RHPA4259 (female; single variant transmission); and 9010 (female; single variant transmission) as described by [2]).Transmission of Founder HIV-1 to Cervical ExplantsIn parallel, we employed the full-length IMC, CH077.t and RHPA.c, which represent the T/F viruses from subjects 700010077 and RHPA4259, respectively [6]. We have previously established that the cellular tropism of Env-IMC closely match that of their respective matched full-length IMC or isolates [6]. The R5-tropic HIV-1BaL (NIH AIDS Research Reference Reagent Program, catalogue #510), isolated from a chronically infected human infant lung, served as another control virus. Virus stocks were prepared essentially as described [4,6]. Briefly, 293T cells were transfected with proviral DNA, medium was changed at 16 hours, and virus stocks harvested at 60 hours. HIV1 BaL was grown in PBMC. All stocks were titered on TZM-bl cells, and infectious units (IU) per ml were determined by betagalactosidase staining for quality assurance. Viral stocks were directly used for inoculation of tissues. TCID50 on 1676428 TZM-bl cells of the different viruses varied from 1×107 to 4.5×107 (for the C/R HIV-1 variants the range was from 2.5 to 4.0 x107 and for T/F HIV-1 variants it was from 1.0 to 4.5×107). Such differences in TCID50 24272870 values measured in one system do not directly translate into consistent differences in virus replication capacity in another system, in this case in tissues from various donors [7]. Furthermore, the observed differences in TCID50 of different viruses are much less than the variability that is seen for replication of a given virus stock in tissues from different donors [5,8].determined by staining with a KC57 FITC labeled anti HIV-1 p24 antibody (Beckman Coulter, Miami, FL).Statistical AnalysesAnalyses were conducted using JMP 9.0 (SAS Institute, Cary, NC). Data were order CP21 analyzed for normality using the Shapiro-Welsh test. When 3 or more groups were compared, we performed an ANOVA with the post-hoc correction of Tukey-kramer Honestly Significant Difference. When data were not normally distributed, we performed a non-parametric multiple comparison with Dunn’s correction for joined ranks. The proportion of successful infection (.100 pg p24) in tissues infected with T/F or C/R viruses were compared using Fishers’ exact test for two group comparisons or the likelihood ratio when successful infection proportions were compared across several groups. In several cases, for the reader’s information, we present both mean 6 SEM and median with IQR. However, in cases of non-normal distribution of the.Ckbones infectious molecular clones (Env-IMC), refered here as “NL-Env.ecto”. To avoid creating chimerism in any reading frames overlapping env, heterologous env sequences encoding the ectodomain were cloned into the NL4-3 backbone, wherein the recombinant viruses express full-length Env [4]. We used three R5-tropic reference HIV-1 variant (NL-SF162.ecto, NL-YU-2.ecto, and NL-BaL.ecto), as well as eight viruses encoding CCR5-utilizing env genes from clade B mucosally transmitted T/F HIV-1 variants, including NL-CH077.ecto, NL-1051.TD12.ecto, NL-1051.C22.ecto, NL-TT31P.2G1.ecto, NL-TT31P.2F10.ecto, NL-SC22.3C2.ecto, NL-RHPA.ecto, and NL-9010.A1.ecto, with env genes derived, respectively, from subjects 700010077 (male; single variant transmission); 1051-12 (female; infected with two related variants); TT31P (female; infected with two related variants); SC22 (female; single variant transmission), RHPA4259 (female; single variant transmission); and 9010 (female; single variant transmission) as described by [2]).Transmission of Founder HIV-1 to Cervical ExplantsIn parallel, we employed the full-length IMC, CH077.t and RHPA.c, which represent the T/F viruses from subjects 700010077 and RHPA4259, respectively [6]. We have previously established that the cellular tropism of Env-IMC closely match that of their respective matched full-length IMC or isolates [6]. The R5-tropic HIV-1BaL (NIH AIDS Research Reference Reagent Program, catalogue #510), isolated from a chronically infected human infant lung, served as another control virus. Virus stocks were prepared essentially as described [4,6]. Briefly, 293T cells were transfected with proviral DNA, medium was changed at 16 hours, and virus stocks harvested at 60 hours. HIV1 BaL was grown in PBMC. All stocks were titered on TZM-bl cells, and infectious units (IU) per ml were determined by betagalactosidase staining for quality assurance. Viral stocks were directly used for inoculation of tissues. TCID50 on 1676428 TZM-bl cells of the different viruses varied from 1×107 to 4.5×107 (for the C/R HIV-1 variants the range was from 2.5 to 4.0 x107 and for T/F HIV-1 variants it was from 1.0 to 4.5×107). Such differences in TCID50 24272870 values measured in one system do not directly translate into consistent differences in virus replication capacity in another system, in this case in tissues from various donors [7]. Furthermore, the observed differences in TCID50 of different viruses are much less than the variability that is seen for replication of a given virus stock in tissues from different donors [5,8].determined by staining with a KC57 FITC labeled anti HIV-1 p24 antibody (Beckman Coulter, Miami, FL).Statistical AnalysesAnalyses were conducted using JMP 9.0 (SAS Institute, Cary, NC). Data were analyzed for normality using the Shapiro-Welsh test. When 3 or more groups were compared, we performed an ANOVA with the post-hoc correction of Tukey-kramer Honestly Significant Difference. When data were not normally distributed, we performed a non-parametric multiple comparison with Dunn’s correction for joined ranks. The proportion of successful infection (.100 pg p24) in tissues infected with T/F or C/R viruses were compared using Fishers’ exact test for two group comparisons or the likelihood ratio when successful infection proportions were compared across several groups. In several cases, for the reader’s information, we present both mean 6 SEM and median with IQR. However, in cases of non-normal distribution of the.
