Assembly and Replication of HIV-1 in T Cells with Low Levels of Phosphatidylinositol-(4,5)-Bisphosphate

199

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCEThe lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Lipid-enveloped viruses harbor a lipid membrane bilayer derived from their host cell during the budding process. This envelope provides the virus stability, protection of its genetic contents, and a reservoir for its transmembrane glycoprotein, which mediates entry into cells (1, 2). The viral lipid envelope may be a viable target for drug development, as particular alterations in the lipid coat or receptor-lipid interaction can inhibit viral entry (3-6). The lipid-dependent budding and egress of some lipid-enveloped viruses have been investigated. For example, it is well established that HIV-1 binds and utilizes 1,2-dioleoyl-sn-glycero-3-phospho-(1=-myo-inositol-4=,5=-bisphosphate) [PI(4,5)P 2 ] enriched in the plasma membrane (PM) inner leaflet for assembly and egress from the cell (7,8). Enteroviruses and flaviviruses use a phosphatidylinositol-4-phosphate [PI(4)P]-enriched organelle to replicate (9), and enteroviruses are packaged into phosphatidylserine (PS)-enriched vesicles, thereby enhancing the efficiency of viral transmission (10). The budding and egres...

“…4A and B). Localization of charge and phosphoinositide reporters and localization of HIV-1 gag, which is dependent upon the presence of PI(4,5)P 2 (7,8), were unchanged ( Fig. 4A and B).…”

Section: Resultsmentioning

confidence: 88%

mentioning

confidence: 99%

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

Adu-Gyamfi

Johnson

Fraser

et al. 2015

199

“…pNL4-3/KFS/398/IRES-Myc-5ptaseIV and pNL4-3/KFS/398/IRESMyc-5ptaseIV⌬1 encode Myc-5ptaseIV and an inactive deletion mutant, Myc-5ptaseIV ⌬1, respectively, following an internal ribosome entry site (IRES) sequence in place of the nef gene, as previously described (76). These plasmids were derived from a parental plasmid, pNL4-3/KFS/398, which has the nef gene sequence replaced with a sequence containing multiple restriction sites derived from plasmid p398-6 (a kind gift from K. T. Jeang) and contains a frameshift mutation (KFS) that disrupts Env expression (77).…”

Section: Cells and Plasmidsmentioning

confidence: 99%

“…These plasmids were derived from a parental plasmid, pNL4-3/KFS/398, which has the nef gene sequence replaced with a sequence containing multiple restriction sites derived from plasmid p398-6 (a kind gift from K. T. Jeang) and contains a frameshift mutation (KFS) that disrupts Env expression (77). pNL4-3/Gag-YFP/ KFS/398/IRES-Myc-5ptaseIV and pNL4-3/Gag-YFP/398/IRES-Myc5ptaseIV⌬1 were constructed from pNL4-3/Gag-YFP by using standard molecular cloning techniques (76). pNL4-3/1GA Gag-YFP (78), pNL4-3/29KT/31KT Gag-YFP (46), pNL4-3/Fyn(10)/⌬MA Gag-YFP (40), pNL4-3/PH/⌬MA Gag-YFP (79), pNL4-3/delNC Gag-YFP (78), pNL4-3/GagLZ-YFP (67), and pNL4-3/Gag-LZ4-YFP (66) were de-scribed previously.…”

Section: Cells and Plasmidsmentioning

confidence: 99%

Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages

Inlora

Chukkapalli

Bedi

et al. 2016

Self Cite

The subcellular sites of HIV-1 assembly, determined by the localization of the structural protein Gag, vary in a cell-type-dependent manner. In T cells and transformed cell lines used as model systems, HIV-1 assembles at the plasma membrane (PM). The binding and localization of HIV-1 Gag to the PM are mediated by the interaction between the matrix (MA) domain, specifically the highly basic region, and a PM-specific acidic phospholipid, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ]. In primary macrophages, prominent accumulation of assembling or assembled particles is found in the virus-containing compartments (VCCs), which largely consist of convoluted invaginations of the PM. To elucidate the molecular mechanism of HIV-1 Gag targeting to the VCCs, we examined the impact of overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P 2 , in primary macrophages. We found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression, suggesting an important role for the MA-PI(4,5)P 2 interaction in HIV-1 assembly in primary macrophages. However, our analysis of HIV-1 Gag derivatives with MA changes showed that this interaction contributes to Gag membrane binding but is dispensable for specific targeting of Gag to the VCCs per se. We further determined that deletion of the NC domain abolishes VCC-specific localization of HIV-1 Gag. Notably, HIV-1 Gag localized efficiently to the VCCs when the NC domain was replaced with a leucine zipper dimerization motif that promotes Gag multimerization. Altogether, our data revealed that targeting of HIV-1 Gag to the VCCs requires NC-dependent multimerization. IMPORTANCEIn T cells and model cell lines, HIV-1 Gag localizes to the PM in a manner dependent on the MA-PI(4,5)P 2 interaction. On the other hand, in primary macrophages, HIV-1 Gag localizes to convoluted intracellular membrane structures termed virus-containing compartments (VCCs). Although these compartments have been known for decades, and despite the implication of viruses in VCCs being involved in virus reservoir maintenance and spread, the viral determinant(s) that promotes Gag targeting to VCCs is unknown. In this study, we found that the MA-PI(4,5)P 2 interaction facilitates efficient Gag membrane binding in macrophages but is not essential for Gag targeting to VCCs. Rather, our results revealed that NC-dependent multimerization promotes VCC targeting. Our findings highlight the differential roles played by MA and NC in HIV-1 Gag membrane binding and targeting and suggest a multimerization-dependent mechanism for Gag trafficking in primary macrophages similar to that for Gag localization to uropods in polarized T cells.

“…Several studies have shown that Vpu expression can be rapidly lost during in vitro culture (22,23). The deletion of vpu from HIV-1 does not greatly diminish the spread of virus in infected T cell lines, implying that viral release is not essential for spread of HIV in cell culture (24).…”

mentioning

confidence: 99%

HIV-1 Vpu Antagonism of Tetherin Inhibits Antibody-Dependent Cellular Cytotoxic Responses by Natural Killer Cells

Alvarez

Hamlin

Monroe

et al. 2014

122

142

The type I interferon-inducible factor tetherin retains virus particles on the surfaces of cells infected with vpu-deficient human immunodeficiency virus type 1 (HIV-1). While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpudeficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating Fc␥RIIIa signaling and enhanced NK cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1-infected primary CD4؉ T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive-patient-derived antibodies. The magnitudes of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surfaces of infected primary CD4 T cells. The affinity of antibody binding to Fc␥RIIIa was also found to be critical in mediating efficient Fc activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibody-mediated clearance of virally infected cells. IMPORTANCEThe ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports the hypothesis that a major function of Vpu is to decrease the recognition of infected cells by anti-HIV antibodies at the cell surface, thereby reducing recognition by antibody-dependent clearance by natural killer cells.