The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells

Abstract

Budding of retroviruses from polarized epithelial Madin–Darby canine kidney cells (MDCK) takes place specifically at the basolateral membrane surface. This sorting event is suspected to require a specific signal harbored by the viral envelope glycoprotein and it was previously shown that, as for most basolateral proteins, the intracytoplasmic domain plays a crucial role in this targeting phenomenon. It is well known that tyrosine‐based motifs are a central element in basolateral targeting signals. In the present study, site‐directed mutagenesis was used to generate conservative or non‐conservative substitutions of each four intracytoplasmic tyrosines of the human immunodeficiency virus (HIV‐1) envelope glycoprotein. This approach revealed that the membrane‐proximal tyrosine is essential to ensure both the basolateral localization of envelope glycoprotein and the basolateral targeting of HIV‐1 virions. Substitutions of the membrane‐proximal tyrosine did not appear to affect incorporation of envelope glycoprotein into the virions, as assayed by virion infectivity and protein content, nor its capability to ensure its role in viral infection, as determined by viral multiplication kinetics. Altogether, these results indicate that the intracytoplasmic domain of the HIV‐1 envelope glycoprotein harbors a unique, tyrosine‐based, basolateral targeting signal. Such a tyrosine‐based targeting signal may play a fundamental role in HIV transmission and pathogenesis.

The Simian Immunodeficiency Virus Envelope Glycoprotein Contains Multiple Signals that Regulate its Cell Surface Expression and Endocytosis

“…Interaction with AP1 would be expected to occur in the trans Golgi network and may direct newly synthesised Env to endosomes. Whether this is required to divert newly synthesised Env to lysosomes (28), thereby providing an additional mechanism to limit Env expression on the cell surface, or plays a more significant role in polarised sorting or recycling of Env remains to be determined (3,33). By contrast to studies in which the HIV-1 Env endocytosis signal was shown to interact with three different m subunits (17), we failed to detect any interaction between a peptide encoding the entire BK28 cytoplasmic domain and AP3 using an optical biosensor.…”

MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes

“…The Env-negative proviral construct harboring a mutation in the matrix protein HXBH10Env-(KpnIfs)⌬16-18 was generated by cloning the SalI-BamHI (HXBH10 nucleotide positions 5372 and 8058, respectively; +1 = site of initiation of transcription) fragment of HXBH10Env-(KpnIfs) into HXBH10⌬16-18; both plasmids have been described elsewhere. 25,37,38 Cell lines and antisera The 293T, COS, HeLa, HeLaCD4 39 and HeLaCD4LTR␤-gal 33 cells were maintained in DMEM supplemented with 5% fetal calf serum (FCS) and 1% streptomycin and penicillin. Human antiserum No.…”

“…162 against HIV proteins has been described elsewhere. 37 The goat antiserum against MuLV Gag (76S000127 and 79S000804) was obtained from Quality Biotech (Biological Carcinogenesis Branch, NCI, Camden, NJ, USA); antiserum raised against MuLV Env (80S00019) was also obtained from Quality Biotech.…”

“…37 Briefly, 10 g of the GagPol encoding plasmids (pHIT60, HXBH10Env-(KpnIfs) or HXBH10Env-(KpnIfs)⌬16-18) were cotransfected with 15 g of one of the following Env encoding plasmid constructs: pHIT456, pSVIIIEnv or pSVIIIEnv713. When pHIT60 was cotransfected with the HIV Env or truncated HIV Env plasmid expression vectors, 5 g of a HIV transactivating protein (Tat) encoding plasmid (SVCMVTat) was added to the DNA mix before transfection, since transcription of both Env genes is driven by the HIV LTR, and their efficient expression is thus dependent on the presence of Tat.…”

“…Viral proteins present in lysed cells or viral pellets were immunoprecipitated with a mix consisting of HIV-positive human, goat anti-MuLV Gag and goat anti-MuLV Env antisera (all used at 1:5000 dilutions) and loaded on to an 11% SDS-PAGE gel as described previously. 37 Labeled proteins were then revealed by autoradiography.…”