Réjean Cantin

The acquisition of host-encoded proteins by nascent HIV-1

Abstract The human immunodeficiency virus type 1 (HIV-1) incorporates several host-derived proteins during the budding process. Here, Michel Tremblay and colleagues review recent studies that indicate that virion-bound host molecules are still functional and are affecting the biological properties of HIV-1. Therefore, these nonviral components should be considered as factors putatively affecting the pathogenesis of HIV-1 infection.

Discrimination between exosomes and HIV-1: Purification of both vesicles from cell-free supernatants ☆

Although enveloped retroviruses bud from the cell surface of T lymphocytes, they use the endocytic pathway and the internal membrane of multivesicular bodies for their assembly and release from macrophages and dendritic cells (DCs). Exosomes, physiological nanoparticles produced by hematopoietic cells, egress from this same pathway and are similar to retroviruses in terms of size, density, the molecules they incorporate and their ability to activate immune cells. Retroviruses are therefore likely to contaminate in vitro preparations of exosomes and vice versa and sucrose gradients are inefficient at separating them. However, we have found that their sedimentation velocities in an iodixanol (Optiprep) velocity gradient are sufficiently different to allow separation and purification of both vesicles. Using acetylcholinesterase as an exosome marker, we demonstrate that Optiprep velocity gradients are very efficient in separating exosomes from HIV-1 particles produced on 293T cells, primary CD4(+) T cells, macrophages or DCs, with exosomes collecting at 8.4-12% iodixanol and HIV-1 at 15.6%. We also show that immunodepletion with an anti-acetylcholinesterase antibody rapidly produces highly purified preparations of HIV-1 or exosomes. These findings have applications in fundamental research on exosomes and/or AIDS, as well as in clinical applications where exosomes are involved, more specifically in tumour therapy or in gene therapy using exosomes generated from DCs genetically modified by transfection with virus.

Plunder and Stowaways: Incorporation of Cellular Proteins by Enveloped Viruses

To be successful and have the opportunity to replicate properly, a virus has to thwart or, just the reverse, boost many systems of the host cell. Given that host cells have evolved to eliminate these hostile parasites, a fierce battle ensues. There is now compelling evidence that enveloped virions released from infected cells will carry the vestiges of this battle both internally and externally. The focus of the present minireview will be the incorporation of the host cellular proteins into or onto the newly formed viruses. The roles of a few of these host cellular proteins have been studied, albeit very recently, because of their implication in the biology of some viruses. But for the vast majority, only the observation of their incorporation has been reported in the scientific literature. The various studies in this research area have been conducted with eukaryotic cells infected primarily with RNA enveloped viruses. Human immunodeficiency virus type 1 (HIV-1) has been the most extensively studied in this respect, but other viruses have also contributed to a better understanding of this intriguing phenomenon. A list of the molecules that have been reported to be embedded in some enveloped viruses is shown in Table ​Table1.1. The processes through which cellular proteins are acquired by viruses are still ill defined. Here we will describe and discuss the roles, or potential roles, that the major cellular proteins found associated with enveloped viruses may play in their life cycles. First, the internally associated host cell molecules will be described, and in a second section, the cell surface constituents found included within the envelopes of different viruses will be introduced. TABLE 1. Host cellular molecules acquired by some enveloped viruses

HIV-1 transcription and virus production are both accentuated by the proinflammatory myeloid-related proteins in human CD4+ T lymphocytes

S100A8, S100A9, and S100A12, collectively known as myeloid-related proteins (MRPs), are highly expressed by the myeloid cell lineage and are found in the extracellular milieu during infections and inflammatory conditions. Recent data showed high levels of MRPs in the serum of HIV type 1 (HIV-1)-infected patients which correlated with disease progression and low CD4+ counts. Therefore, we set out to investigate the effect of MRPs on HIV-1 replication. We observed a 4- to 5-fold induction of virus production in J1.1, a human T lymphoid cell line latently infected with HIV-1, following treatment with MRPs. Using luciferase-based reporter gene assays, we demonstrated that MRPs induce a dose- and time-dependent activation of the HIV-1 long terminal repeat promoter region that could be blocked by specific anti-MRP polyclonal Abs and by physical denaturation of these proteins. The MRP-mediated induction was acting through the HIV-1 enhancer sequence and was dependent upon NF-κB activity. These latter results were also confirmed by EMSA experiments conducted in Jurkat cells and freshly isolated PBMCs. In conclusion, we demonstrate that MRPs induce HIV-1 transcriptional activity and viral replication in infected CD4+ T-lymphocytes at concentrations similar to those found in the serum of HIV-1-infected patients.

The importance of virus-associated host ICAM-1 in human immunodeficiency virus type 1 dissemination depends on the cellular context

The primary objective of this study was to define whether the nature of virion‐bound host cell membrane proteins influenced the process of human immunodeficiency virus 1 (HIV‐1) capture and transmission. We pulsed cells of monocytoid lineage (established and primary) and CD4‐ negative epithelial cells transiently expressing DC‐SIGN or LFA‐1 with isogenic HIV‐1 particles either devoid or bearing host‐derived ICAM‐1 or ICAM‐3 before incubation with an indicator cell line. To our surprise, the ICAM‐1/LFA‐1 association was a more efficient transmission factor than the combined gp120/DC‐SIGN and ICAM‐3/DC‐SIGN interactions. The involvement of the association between virus‐bound ICAM‐1 and its natural ligand LFA‐1 in virus binding and carriage was confirmed when using more physiological cellular targets, i.e., human lymphoid tissues cultured ex vivo. However, the contribution of virus‐anchored host ICAM‐1 to the process of retention and transmission of HIV‐1 could not be confirmed when using primary human cells of macrophage/dendritic lineage as transmitter cells and autologous CD4+ T lymphocytes as targets. Altogether these data underscore the complexity of factors participating in virus‐cell contact and efficient dissemination of HIV‐1 to target cells.

A novel virus capture assay reveals a differential acquisition of host HLA-DR by clinical isolates of human immunodeficiency virus type 1 expanded in primary human cells depending on the nature of producing cells and the donor source.

Previous findings indicated that HLA-DR is probably one of the most abundant cellular constituents incorporated within the human immunodeficiency virus type 1 (HIV-1) envelope. Given that the life-cycle of HIV-1 has been reported to be modulated by virion-bound host HLA-DR, an improved version of a virus capture technique was developed to assess the degree of HLA-DR incorporation in several clinical isolates of HIV-1 derived from primary human peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDM). Analysis of virus stocks purified from PBMCs and MDM indicated that primary isolates of HIV-1 bearing distinct tropism (i.e. T-, macrophage-, and dual-tropic) all incorporate host cell membrane HLA-DR protein. The amount of incorporated HLA-DR varies among the primary HIV-1 isolates tested. Propagation of some clinical HIV-1 isolates in either autologous PBMCs or MDM resulted in differential incorporation of virion-bound cellular HLA-DR depending on the nature of the virus producer cells. Differences in the degree of HLA-DR incorporation were also noticed when macrophage-tropic isolates of HIV-1 were produced in MDM from different donors. Altogether these data show that the efficiency of HLA-DR incorporation into the envelope of primary isolates of HIV-1 is a multifactorial phenomenon since it is affected by the virus isolate itself, the nature of host cells (i.e. PBMCs or MDM) and the donor source.

Involvement of Src and Syk Tyrosine Kinases in HIV-1 Transfer from Dendritic Cells to CD4+ T Lymphocytes

Dendritic cells (DCs) are considered as key mediators of the early events in HIV-1 infection at mucosal sites. Although several aspects of the complex interactions between DCs and HIV-1 have been elucidated, there are still basic questions that remain to be answered about DCs/HIV-1 interplay. In this study, we examined the contribution of nonreceptor TKs in the known ability of DCs to efficiently transfer HIV-1 to CD4+ T cells in trans. Experiments performed with specific inhibitors of Src and Syk family members indicate that these tyrosine kinases (TKs) are participating to HIV-1 transfer from immature monocyte-derived DCs (IM-MDDCs) to autologous CD4+ T cells. Experiments with IM-MDDCs transfected with small interfering RNAs targeting Lyn and Syk confirmed the importance of these nonreceptor TKs in HIV-1 transmission. The Src- and Syk-mediated effect on virus transfer was linked with infection of IM-MDDCs in cis-as monitored by quantifying integrated viral DNA and de novo virus production. The process of HIV-1 transmission from IM-MDDCs to CD4+ T cells was unaffected following treatment with protein kinase C and protein kinase A inhibitors. These data suggest that Src and Syk TKs play a functional role in productive HIV-1 infection of IM-MDDCs. Additional work is needed to facilitate our comprehension of the various mechanisms underlying the exact contribution of Src and Syk TKs to this phenomenon.

New Insights into the Functionality of a Virion-Anchored Host Cell Membrane Protein: CD28 Versus HIV Type 1

It is now well established that the HIV type 1 (HIV-1) incorporates a vast array of host-encoded molecules in its envelope during the budding process. Interestingly, it was demonstrated that the attachment process is accentuated by supplementary interactions between virion-anchored host molecules and their cognate ligands. Such an enhancement of the viral attachment process was found to result in an increase of infectivity for both T and macrophage-tropic strains of HIV-1. Given that previous work indicates that HIV-1 is budding at the site of cell-to-cell contact, a location rich in the costimulatory CD28 glycoprotein, we investigated whether CD28 could be efficiently acquired by HIV-1. We have been able to generate progeny viruses bearing or not bearing on their surfaces host-derived CD28 using our previously described transient transfection and expression system. The physical presence of CD28 was found to markedly increase virus infectivity in a CD28/B7-dependent manner following infection of two human lymphoid cell lines expressing high levels of surface B7-1/B7-2, two natural ligands of CD28. The physiological significance of CD28 incorporation was provided by the observation that an anti-CD28 Ab decreased replication in primary human mononuclear cells of clinical isolates of HIV-1 propagated in such cells. A virus precipitation assay revealed that M-, T-, and dual-tropic clinical strains of HIV-1 produced in primary human mononuclear cells do indeed incorporate CD28. These results show for the first time that HIV-1 can incorporate CD28 and the acquisition of this specific host surface glycoprotein modulates the virus life cycle.

Human immunodeficiency virus type 1 replication in dendritic cell-T-cell cocultures is increased upon incorporation of host LFA-1 due to higher levels of virus production in immature dendritic cells

ABSTRACT Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4+ T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.

DC-SIGN-Mediated Transfer of HIV-1 Is Compromised by the Ability of Leishmania infantum to Exploit DC-SIGN as a Ligand

DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) binds human immunodeficiency virus type 1 (HIV-1) and facilitates transfer of virus to permissive cells. Leishmania parasites also exploit DC-SIGN as a receptor. Here, we report that transfer of HIV-1 to target cells is markedly reduced when DC-SIGN(+) cells are preincubated with Leishmania amastigotes before pulsing with virions. Moreover, binding of HIV-1 to DC-SIGN(+) cells is diminished by the presence of Leishmania amastigotes. Our findings provide novel insight into the complex interactions between HIV-1 and Leishmania parasites. The ability of both HIV-1 and Leishmania parasites to bind to the same cell-surface constituent to gain entry into dendritic cells might have an impact on the immunological and pathological events associated with HIV-1 infection.