Second site reversion of HIV-1 envelope protein baseplate mutations maps to the matrix protein.

The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.

A nanobody interaction with SARS-COV-2 Spike allows the versatile targeting of lentivirus vectors.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

A Nanobody Interaction with SARS-CoV-2 Spike Allows the Versatile Targeting of Lentivirus Vectors.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor binding domain to direct lentivirus infection of Spike-expressing cells. Using three different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. The targeting is dependent on the fusion function of Spike, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion, and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections. IMPORTANCE: We have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein, and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion, and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

Analysis of HIV-1 envelope cytoplasmic tail effects on viral replication.

Trimers of the HIV-1 envelope (Env) protein perform receptor binding and virus-cell fusion functions during the virus life cycle. The cytoplasmic tail (CT) of Env forms an unusual baseplate structure, and is palmitoylated, rich in arginines, carries trafficking motifs, binds cholesterol, and interacts with host proteins. To dissect CT activities, we examined a panel of Env variants, including CT truncations, mutations, and an extension. We found that whereas all variants could replicate in permissive cells, viruses with CT truncations or baseplate mutations were defective in restrictive cells. We also identified a determinant in HIV-1 amphotericin sensitivity, and characterized variants that escape amphotericin inhibition via viral protease-mediated CT cleavage. Results additionally showed that full-length, his tagged Env can oligomerize and be co-assembled with CT truncations that delete portions of the baseplate, host protein binding sites, and trafficking signals. Our observations illuminate novel aspects of HIV-1 CT structure, interactions, and functions.

Capsid-specific nanobody effects on HIV-1 assembly and infectivity.

The capsid (CA) domain of the HIV-1 precursor Gag (PrGag) protein plays multiple roles in HIV-1 replication, and is central to the assembly of immature virions, and mature virus cores. CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs). We have investigated the interactions of CA with anti-CA nanobodies, which derive from the antigen recognition regions of camelid heavy chain-only antibodies. The one CA NTD-specific and two CTD-specific nanobodies we analyzed proved sensitive and specific HIV-1 CA detection reagents in immunoassays. When co-expressed with HIV-1 Gag proteins in cells, the NTD-specific nanobody was efficiently assembled into virions and did not perturb virus assembly. In contrast, the two CTD-specific nanobodies reduced PrGag processing, virus release and HIV-1 infectivity. Our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1.