Protein-modifying Variants Increase Viral Capture and Transfer to T cells DC-SIGN molecules on the cell surface enhance HIV-1 infection by capturing virions and transmitting them to CD4 + T-lymphocytes [14, 15] . The neck region, encoded by exon 4, is important for efficient binding to HIV-1 [35] . We hypothesized that the exon 4 protein-modifying variants associated with IU HIV-1 infection could affect the interaction between DC-SIGN and HIV-1. To assess viral capture, exon 4 from the DC-SIGN expression vector was replaced by exon 4 from infants carrying WT, 242V (designated as L242V) or 198Q, 214D and 221Q (designated as R198Q) variants ( Figure 3A ). Raji cells do not express endogenous DC-SIGN and allowed us to investigate the net impact of exon 4 mutations on DC-SIGN HIV-1 affinity. Raji cells ( Figure 3B) were stably transfected and cell lines grown from a single clone. Since viral capture is influenced by cell-surface expression of DC-SIGN [35] , we selected cell lines with similar baseline DC-SIGN surface expression ( Figure 3B ). The stable Raji transfectants were pulsed with equal amount of R5 tropic HIV-1 HXBRU-ADA or HIV-1 JRCSF strains extensively washed to remove the unbound virus, and then lysed. The parental Raji cells were used as controls. The number of virions used was not saturating since capture increased in a dosedependent manner ( Figure S2A ). Interestingly, DC-SIGN L242V and R198Q variants were more efficient at capturing viral particles than WT ( Figure 3C ). HIV-1 capture on the Raji transfectants was stable over time ( Figure S2B ) and dependent on DC-SIGN interaction since the capture was reduced to background levels following incubation with DC-SIGN antibody (AZN-D1) or mannan ( Figure 3C ). Similar results were obtained when cells were pulsed with HIV-1 JRCSF strain ( Figure S2C ). To investigate whether DC-SIGN exon 4 mutations could also enhance cell transmission of HIV-1, we co-cultivated activated primary human CD4 + T lymphocytes with HIV-1 pulsed Raji transfectants. Transmission was quantified by measuring HIV-1 p24 in the supernatants after 5 days. The DC-SIGN variants significantly increased viral transmission to CD4 + T-lymphocytes ( Figure 3D ). Transmission was dependent on DC-SIGN expression since Raji cells or transfectants pre-incubated with DC-SIGN antibody failed to transmit HIV-1 to CD4 + T lymphocytes. Moreover, cell infection was not due to viral particles shed into the supernatant since virus was undetectable in the absence of CD4 + T lymphocytes ( Figure 3D) . Thus, the DC-SIGN neck region variants associated with IU HIV-1 infection enhanced both the capture of HIV-1 by DC-SIGN and its subsequent transmission to the CD4 + T lymphocytes.