Abstract
The CRISPR technology not only can knock out target genes by using the RNA-guided Cas9 nuclease but also can activate their expression when a nuclease-deficient Cas9 (dCas9) is employed. Using the latter function, we here show the effect of the CRISPR-mediated pinpoint activation of endogenous expression of BST-2 (also known as tetherin), a virus restriction factor with a broad antiviral spectrum. Single-guide RNA (sgRNA) sequences targeting the BST-2 promoter were selected by promoter assays. Potential sgRNAs and dCas9 fused to the VP64 transactivation domain, along with an accessory transcriptional activator complex, were introduced into cells by lentiviral transduction. Increased expression of BST-2 mRNA in transduced cells was confirmed by real-time RT-PCR. Cells in which BST-2 expression was highly enhanced showed the effective inhibition of HIV-1 production and replication even in the presence of the viral antagonist Vpu against BST-2. These findings confirm that the physiological stoichiometry between host restriction factors and viral antagonists may determine the outcome of the battle with viruses.
Highlights
In the mid ‘90 s, the requirements of the so-called “accessory” HIV proteins sthat had been long known to be nonessential for viral replication in in vitro culture were reported to depend on the cell-types used for infection experiments[1,2,3,4,5,6]
To identify the best single guide RNA (sgRNA) target sequences in the BST-2 promoter, we cloned its promoter into a firefly luciferase reporter plasmid and cotransfected HeLa cells with this promoter-indicator plasmid together with plasmids expressing sgRNA appended to the phage MS2 RNA stem loop, as well as dCas[9] fused to the herpes simplex virus transcription factor VP16 minimal activation domain termed VP64, and the transcription factor fusion protein that is the NF-kB trans-activating subunit p65 with the activation domain from the human heat-shock factor 1 fused to the phage MS2 coat protein (MS2-p65-HSF1), and we performed luciferase assays
Host restriction factors that are able to inhibit HIV infection are normally counteracted by HIV accessory proteins that act as viral antagonists
Summary
In the mid ‘90 s, the requirements of the so-called “accessory” (or “auxiliary”) HIV proteins sthat had been long known to be nonessential for viral replication in in vitro culture were reported to depend on the cell-types used for infection experiments[1,2,3,4,5,6]. The transmembrane protein BST-2 potently acts by tethering to HIV particles present on the surface of virus-producing cells This restriction factor is very peculiar in that, unlike the other three factors that inhibit retroviral infections, BST-2 displays broad-spectrum activity against a variety of enveloped viruses as well as retroviruses, e.g., Marburg virus[17], Lassa virus[17,18], Ebola virus[19,20], Sendai virus[21], influenza virus[22,23], herpes simplex virus 124, Kaposi’s sarcoma-associated herpesvirus[25], vesicular stomatitis virus[26], chikungunya virus[27], SARS corona virus[28], hepatitis B virus[29], and hepatitis C virus[30], human parainfluenza virus[31], almost all of which harbor different viral antagonists against BST-2. We attempt to enhance the endogenous expression of BST-2 to observe whether the CRISPR-based pinpoint activation of BST-2 expression is able to confer cells with inhibitory activity on HIV-1 virion production, even in the presence of the viral antagonist Vpu that counteracts BST-2 by binding and downregulating the restriction factor[37]
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