Lentivirus-based vectors derived from human immunodeficiency viruses type 1 and 2 (HIV-1 and 2) are widely used tools in research and may also be utilized in clinical settings. Like their parental virions, they are known to depend on the cellular machinery for successful gene delivery and integration. While most of the studies on cellular proteomic and transcriptomic changes have focused on the late phase of the transduction, studies of those changes in early time-points, especially in the case of HIV-2 based vectors, are widely lacking. Using second generation HIV-1 and 2 vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviral vectors, we transduced HEK-293T human embryonic kidney cells and carried out transcriptomic profiling at 0 and 2 h time points, with accompanying proteomic analysis at 2 h following transduction. Significant variations were observed in gene expression profile between HIV-1 and HIV-2 transduced samples. Thrombospondin 1 (THBS1), collagens (COL1A2, COL3A1), and eukaryotic translation factors (EIF3CL) in addition to various genes coding for long non-coding RNA (lncRNA) were significantly upregulated 2 h after HIV-2 transduction compared to HIV-1. Label-free quantification mass spectrometry (MS) indicated that seven proteins involved in RNA binding, mRNA transport, and chaperoning were significantly downregulated. The identification of cellular protein targets of lentiviral vectors and their effect on the cellular transcriptome will undoubtedly shed more light on their complex life cycle and may be utilized against infection by their parental lentiviruses. Furthermore, characterizing the early phase of HIV-2 infection may aid in the understanding of its pathomechanism and long incubation period.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19). The spike protein (S) of SARS-CoV-2 plays a crucial role in mediating viral infectivity; hence, in an extensive effort to curb the pandemic, many urgently approved vaccines rely on the expression of the S protein, aiming to induce a humoral and cellular response to protect against the infection. Given the very limited information about the effects of intracellular expression of the S protein in host cells, we aimed to characterize the early cellular transcriptomic changes induced by expression of the S protein in THP-1-derived macrophage-like cells. Results showed that a wide variety of genes were differentially expressed, products of which are mainly involved in cell adhesion, homeostasis, and most notably, antiviral and immune responses, depicted by significant downregulation of protocadherins and type I alpha interferons (IFNAs). While initially, the levels of IFNAs were higher in the medium of S protein expressing cells, the downregulation observed on the transcriptomic level might have been reflected by no further increase of IFNA cytokines beyond the 5 h time-point, compared to the mock control. Our study highlights the intrinsic pathogenic role of the S protein and sheds some light on the potential drawbacks of its utilization in the context of vaccination strategies.
Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) are the causative agents of the acquired immunodeficiency syndrome (AIDS). While both viruses share a similar structural and genomic organization, a difference in replication dynamics and the clinical course of infection is evident between the two. Patients dually infected were shown to have lower viral loads and generally a slower rate of progression to AIDS than those who are mono-infected. While the roles of the unique accessory proteins have been studied in detail for HIV-1, those of HIV-2, including viral protein X (Vpx), remain largely uncharacterized. In our previous experiments, Vpx of HIV-2 was found to be involved in decreasing the infectivity of HIV-1 in dual infection cell culture assays. We set out to elucidate the function of this accessory protein, identifying protein–protein interactions of HIV-2 Vpx with cellular and possibly HIV-1 proteins in dual infection, using in-vitro proteomics techniques and proximity ligation assays. Results showed that wild-type Vpx interacted with many cellular proteins involved in splicing, packaging of pre-mRNA, nuclear export, and translation. Of particular interest was the interaction between HIV-2 Vpx and the pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15, which is required for HIV-1 viral DNA synthesis, and the eukaryotic translation initiation factor 2 subunit 3 (EIF2S3), involved in the early steps of protein synthesis. Additionally, Vpx was found to interact directly with the cellular transcriptional repressor C-Terminal Binding Protein 2 (CTBP-2). Moreover, Vpx was shown to hinder the function of HIV-1 reverse transcriptase in in-vitro assays. These findings shed light on the functions of this accessory protein and add to our understanding of the replication dynamics of HIV-2 and its role in dual infection.
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