Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

2014

Viruses

The lentiviral envelope glycoproteins (Env) mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV) infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively) are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles.

Section: Current Knowledge On the Process Of Fiv Env Incorporationmentioning

confidence: 99%

Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

2014

Viruses

“…31 After infection, the cells were washed twice with DMEM and then transfected with the SU-HA, SU DV3 -HA, and SU HV3 -HA plasmids using Lipofectamine 2000 (Invitrogen) as previously described. 25,32 Thirty hours postinfection, culture supernatants were filtered through 0.45-lm-pore-size filters (Corning) and the amounts of the three different SU-HA glycoproteins present in the culture media were normalized by western blotting using the anti-HA MAb.…”

Section: Expression Of the Su-ha Glycoproteinsmentioning

confidence: 99%

“…To determine the levels at which the SU WT -HA and SU HV3 -HA bind to the surface of HeLa cells, we also performed cellular enzyme-linked immunosorbent assays (cellular ELISA) similar to those previously described. 32 Triplicate samples corresponding to 3 • 10 5 HeLa cells were first incubated at 37°C for 1 h with supernatants containing normalized amounts of the two different SU-HA glycoproteins, washed three times with ice-cold PBS, and resuspended in 50 ll of ELISA buffer (PBS containing 0.4% bovine serum albumin and 0.1% sodium azide). Cells were incubated for 1 h at 4°C with the anti-HA MAb conjugated to horseradish peroxidase followed by three washes with ice-cold ELISA buffer.…”

Section: Cellular Elisamentioning

confidence: 99%

“…Coculture was continued for 48 h, after which cells were stained for b-galactosidase activity and scored for syncytia formation as previously described. 25,32 Cell-to-cell fusion was quantitated as the total number of blue syncytia per well by first counting their number in 20 nonoverlapping fields. The average number of syncytia per field was multiplied by the total number of fields per well and the result was referred to that obtained for the wild-type FIV Env protein.…”

Section: Cell-to-cell Fusion Assaysmentioning

confidence: 99%

“…shown that cellular ELISA can successfully be used to determine the relative levels at the cell surface of wild-type and mutant FIV Env proteins. 32 HeLa cells were incubated with SU WT -HA, SU HV3 -HA, or culture medium from mocktransfected cells as negative control, washed, and incubated with the anti-HA MAb conjugated to peroxidase. After washing the cells, color was developed and quantitated as described in Materials and Methods.…”

Section: Figmentioning

confidence: 99%

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Replacement of the V3 Domain in the Surface Subunit of the Feline Immunodeficiency Virus Envelope Glycoprotein with the Equivalent Region of a T Cell-Tropic Human Immunodeficiency Virus Type 1 Results in a Chimeric Surface Protein That Efficiently Binds to CXCR4

Falcon

AIDS Research and Human Retroviruses

2014

Self Cite

Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain

2016

Arch Virol

The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding. Of note, there are two contiguous and highly conserved potential N-glycosylation sites ((418)NST(420) and (422)NLT(424)) located at the C-terminal side of the V3 domain. We therefore decided to study the relevance for Env functions of these N-glycosylation motifs and found that disruption of both of them by introducing the N418Q/N422Q double amino acid substitution drastically impairs Env processing into the SU and TM subunits. Moreover, the simultaneous mutation of these N-glycosylation sites prevents Env incorporation into virions and Env-mediated cell-to-cell fusion. Notably, a recombinant soluble version of the SU glycoprotein carrying the double amino acid replacement N418Q/N422Q at the V3 C-terminal side binds to CXCR4 with an efficiency similar to that of wild-type SU.