Loss of a Conserved N-Linked Glycosylation Site in the Simian Immunodeficiency Virus Envelope Glycoprotein V2 Region Enhances Macrophage Tropism by Increasing CD4-Independent Cell-to-Cell Transmission

Abstract

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains differ in their capacity to replicate in macrophages, but mechanisms underlying these differences are not fully understood. Here, we identify a highly conserved Nlinked glycosylation site (N173 in SIV, corresponding to N160 in HIV) in the V2 region of the SIV envelope glycoprotein (Env) as a novel determinant of macrophage tropism and characterize mechanisms underlying this phenotype. Loss of the N173 glycosylation site in the non-macrophage-tropic SIVmac239 by introducing an N173Q mutation enhanced viral replication and multinucleated giant cell formation upon infection of rhesus macrophages, while the addition of N173 to SIVmac251 had the opposite effect. The removal of N173 in SIVmac239 enhanced CD4-independent cell-to-cell transmission to CCR5-expressing cells. SIVmac239 with N173Q mediated CD4-independent cell-cell fusion but could not infect CD4-negative cells in single-round infections. Thus, CD4-independent phenotypes were detected only in the context of cell-to-cell contact. Similar results were obtained in SIVmac251 with and without N173. N173 decreased the neutralization sensitivity of SIVmac251 but had no effect on the neutralization sensitivity of SIVmac239. The N173Q mutation had no effect on SIVmac239 binding to CD4 in Biacore assays, coimmunoprecipitation assays, and enzyme-linked immunosorbent assays (ELISAs). These findings suggest that the loss of the N173 N-linked glycosylation site increases SIVmac239 replication in macrophages by enhancing CD4-independent cell-to-cell virus transmission through CCR5-mediated fusion. This mechanism may facilitate the escape of macrophage-tropic viruses from neutralizing antibodies while promoting spreading infection by these viruses in vivo. IMPORTANCEIn this study, we identify a genetic determinant in the viral envelope (N173) that increases replication and spreading infection of SIV strains in macrophages by enhancing cell-to-cell virus transmission. This effect is explained by a novel mechanism involving increased cell-to-cell fusion in the absence of CD4, the primary receptor that normally mediates virus entry. The same genetic determinant also affects the sensitivity of these viruses to inhibition by neutralizing antibodies. Most macrophage-tropic HIV/ SIV strains are known to be neutralization sensitive. Together, these findings suggest that this efficient mode of virus transmission may facilitate the escape of macrophage-tropic viruses from neutralizing antibodies while promoting spreading infection by these viruses to cells expressing little or no CD4 in vivo. Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains differ in their ability to infect and replicate in macrophages. Macrophage tropism is determined primarily by the envelope glycoprotein (Env), which forms trimers composed of three noncovalently bound heterodimers of gp120 and gp41 subunits. The gp120 exterior subunit interacts with CD4 and a coreceptor, mainly CCR5 (1, 2),...

Derivation and Characterization of a CD4-Independent, Non-CD4-Tropic Simian Immunodeficiency Virus

“…Because CD4-independent viruses are characteristically neutralization sensitive, it is likely that they are strongly selected against during typical pathogenic infection (32,48,49,97). Recent publications by Yen et al have suggested that CD4-independent entry serves as a mechanism of cell-cell viral spread in tissue macrophages, which is more efficient and can shield virus from neutralizing antibodies than cell-free transmission, thereby allowing CD4-independent variants to circulate in compartments such as the brain (59,60). Interestingly, primary isolates of HIV-2, which is less pathogenic than HIV-1 (reviewed in reference 98), have been reported to exhibit CD4 independence in vitro (61,99).…”

“…For one neuropathic SIV isolate, its ability to cause AIDS encephalopathy in macaques correlated with the infection of brain-derived endothelial cells that expressed CCR5 but not CD4 (58). In addition, CD4-independent infection has been proposed to contribute to SIV infection of macrophages in the context of cell-to-cell transmission (59,60). Of note, viruses that are CD4 independent typically retain their CD4 binding site and the ability to engage CD4, if present (30,34,58,61,62), and have been shown to exhibit faster fusion kinetics in the presence of CD4 than CD4-dependent viruses (63).…”

HIV-1 Envelope Glycan Moieties Modulate HIV-1 Transmission

“…In contrast, recombinant gp120 displays extensive glycan processing by cellular glycosylation enzymes, resulting in glycans composed of Ͼ70% complex glycans (26,27). Studies of HIV-1 glycosylation have focused mainly on the Env structure and surface exposure and their effect on antigenicity, immunogenicity, and antibody neutralization (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) through mutation of the position of glycosylation, rather than modification of the type of glycans. Studies of the effect of glycosylation on HIV-1 infectivity and carbohydrate and receptor binding have also utilized deletion of the potential N-linked glycosylation sites (PNGSs) (15)(16)(17)(18)28).…”

“…N-Glycans include three basic structures, namely, high-mannose (i.e., oligomannose), hybrid, and complex glycans, all of which are present on HIV-1 Env (4-7). Importantly, N-glycans shield the Env backbone and have been shown to play key roles in determining the Env structure, epitope exposure, and, consequently, antigenicity, immunogenicity, antibody neutralization, infectivity, and carbohydrate and receptor binding (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

“…N-Glycans include three basic structures, namely, high-mannose (i.e., oligomannose), hybrid, and complex glycans, all of which are present on HIV-1 Env (4-7). Importantly, N-glycans shield the Env backbone and have been shown to play key roles in determining the Env structure, epitope exposure, and, consequently, antigenicity, immunogenicity, antibody neutralization, infectivity, and carbohydrate and receptor binding (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).The position of N-linked glycans on glycoproteins is genetically encoded, whereas the types of glycans at a given Asn in the consensus Asn-X-Ser/Thr-X sequence (where X is not Pro) are determined by the glycan branching processes in the cell endoplasmic reticulum and Golgi apparatus (21). However, some glycoproteins, including HIV-1 gp120, demonstrate protein-specific glycosylation (22)(23)(24)(25).…”