An amphipathic sequence in the cytoplasmic tail of HIV-1 Env alters cell tropism and modulates viral receptor specificity

Abstract

Summary. -The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from a CD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying a truncated Env (CT84). To determine whether a specific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared a series of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha-helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred a switch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.Keywords: HIV-1; Env glycoprotein; gp41; gp120; cytoplasmic tail truncation; alpha-helical LLP2 sequence; CD4, CD8; receptor specificity E-mail: avzorov@emory.edu; phone: +1-404-727-3228. Abbreviations: CT = cytoplasmic tail; FL = full-length; HIV-1 = human immunodeficiency virus type 1; LLP-1,-2,-3 = conserved alpha-helical "lentivirus lytic peptide" domains; SP = signal peptide sequence; SIV = simian immunodeficiency virus; SU(s) = surface subunit(s); TM = transmembrane protein; WT = wild type of the Env protein (Vzorov and

“Cytoplasmic domain effects on exposure of co-receptor-binding sites of HIV-1 Env”

“…Our previous results indicate that the CT84 domain determines CD8-receptor specificity and that the full-length CT domain determines the CD4-receptor specificity of 92UG046 Env [29]. In contrast, CT17 truncation generates defective Env proteins [2, 27, 29]. To compare the infectivity of virions pseudotyped with chimeric Env containing FL or truncated CT domains, we used TZM-bl cells, which stably express large amounts of CD4 and CCR5 and constitutively express CXCR4.…”

“…Plasmid pBlueSF162 containing the SF162 env gene (accession number EU123924) was provided by Sang-Moo Kang (Georgia State University, Atlanta GA). Plasmids containing the 92UG046 env genes (accession number AY623600) with different CT domain sequences were described before [29]. Plasmid vector pCAGGS (kindly provided by Dr. Y. Kawaoka) containing a CMV/β-actin chimera promoter was used to express Env constructs.…”

“…After 3 days, supernatants were clarified by centrifugation at 3500 rpm for 15 min and analyzed by titration in TZM-bl cells or purified as described [29]. Briefly, concentrated stocks of pseudotyped virions were prepared by filtration through a 0.45-µm syringe filter and pelleting by centrifugation and stored at −80 °C until use.…”

“…To compare infectivity of virus stocks by titration, two methods were compared: using similar amounts of p24 for input and using amounts of virus that give a similar infectious index (IU/ng), which is the ratio between the number of copies of proviral DNA and core antigen, thereby taking both of these characteristics into consideration [27, 29]. …”

“…A conformational change of the Env protein is triggered either by receptor binding or by inside-out signaling from the CT domain [10, 13, 17, 28, 33]. CT modifications can affect the conformation of the external domain of gp41 on the cell surface [24] and may enhance exposure of receptor-binding segments in gp120 [29]. …”