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Background:  The incorporation of envelope glycoprotein onto budding virions is an important step during assembly of HIV-1 in infected cells. The cytoplasmic domain of envelope protein (gp41) has been shown to carry determinants important in envelope incorporation onto virus particles. Two related envelope genes amplified by PCR from lymph node tissue of an AIDS patient conferred cell:cell fusion but were inefficiently assembled onto virions. Here we have analyzed these envelopes for their assembly defects.

Methods:  The envelope genes were subcloned into an envelope expression vector (pSVIIIenv) and pseudotype virus was produced by co-transfection of pSVIIIenv and pNL43env^- in 293T cells. Specific point mutations were introduced in the envelope gene by PCR. Single-round infectivity assays were done on CD4⁺ CCR5⁺ GHOST cells. Cell-cell fusion assays were done by mixing env⁺ 293T cells and target cells, e.g., GHOST/CCR5 cells. Envelope incorporation onto virions was assessed by Western blot of filtered and pelleted virions.

Results:  The patient envelopes expressed on 293T cells induced cell:cell fusion of CD4⁺CCR5⁺ cells. Pseudotype virions harvested from 293T cells co-transfected with patient env⁺pSVIIIenv and pNL43env^- conferred only very low levels of infectivity on CD4⁺ CCR5⁺ GHOST cells. The low infectivity correlated with minimal envelope incorporation onto virions as determined by Western blot. Sequencing and mutagenesis analyses showed critical determinants mapped to a single R787H substitution in a region of the gp41 cytoplasmic domain, previously implicated in gag:env interactions. Reversion of the substitution restored envelope assembly onto virions and infectivity. However, when the same substitution (R787H) was introduced into the NL43 envelope, no effect on assembly or infectivity was observed.

Conclusions:  These results indicate that R787 in the gp41 cytoplasmic domain is an important determinant in envelope assembly but can be complemented by unknown determinant/s present in the NL43 envelope.

Keywords: HIV-1; Assembly; gp41