CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi’s Sarcoma-Associated Herpesvirus-Infected Cells

Brackett, Kevin; Mungale, Ameera; Lopez-Isidro, Mary; Proctor, Duncan A.; Najarro, Guillermo; Arias, Carolina

doi:10.3390/v13050783

Cited by 8 publications

(8 citation statements)

References 52 publications

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“…CRISPR/Cas also has the advantage that multiple sites on the viral genome can be targeted by increasing the number of gRNAs, rather than needing to express two or more enzymes. CRISPR-based genome editing been used to target viruses including hepatitis B virus (HBV) [85,86], human immunodeficiency virus (HIV) [87,88], and several herpesviruses [89] such as EBV [90], CMV [91], Kaposi's Sarcoma Virus [92], and HSV-1 [31][32][33]. In the case of EBV, antiviral gene editing has targeted both the viral genome [90] as well as key cellular components required for EBV replication [93].…”

Section: Discussionmentioning

confidence: 99%

Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

Yee

Goldstein

et al. 2022

Viruses

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Varicella Zoster Virus (VZV) causes Herpes Zoster (HZ), a common debilitating and complicated disease affecting up to a third of unvaccinated populations. Novel antiviral treatments for VZV reactivation and HZ are still in need. Here, we evaluated the potential of targeting the replicating and reactivating VZV genome using Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 nucleases (CRISPR/Cas9) delivered by adeno-associated virus (AAV) vectors. After AAV serotype and guide RNA (gRNA) optimization, we report that a single treatment with AAV2-expressing Staphylococcus aureus CRISPR/Cas9 (saCas9) with gRNA to the duplicated and essential VZV genes ORF62/71 (AAV2-62gRsaCas9) greatly reduced VZV progeny yield and cell-to-cell spread in representative epithelial cells and in lytically infected human embryonic stem cell (hESC)-derived neurons. In contrast, AAV2-62gRsaCas9 did not reduce the replication of a recombinant virus mutated in the ORF62 targeted sequence, establishing that antiviral effects were a consequence of VZV-genome targeting. Delivery to latently infected and reactivation-induced neuron cultures also greatly reduced infectious-virus production. These results demonstrate the potential of AAV-delivered genome editors to limit VZV productive replication in epithelial cells, infected human neurons, and upon reactivation. The approach could be developed into a strategy for the treatment of VZV disease and virus spread in HZ.

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Section: Discussionmentioning

confidence: 99%

Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

Yee

Goldstein

et al. 2022

Viruses

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“…Viral particles were concentrated 5-fold using a regenerated cellulose centrifugal filter unit with a 100k MW cut-off (Amicon Ultracel 100k). The resulting lentivirus stock was used to transduce iSLK.219-dCas9-KRAB cells by spinoculation 37 . Transduced iSLK.219 cells were maintained in 10 μg/mL of puromycin and were selected for BFP+/sgRNA+ expression by FACS in a Sony SH800 instrument.…”

Section: Induction and Assessment Of Kshv Reactivation And Replicationmentioning

confidence: 99%

BiP/GRP78 is a pro-viral factor for diverse dsDNA viruses that promotes the survival and proliferation of cells upon KSHV infection

Najarro,

Brackett,

Woosley

et al. 2023

Preprint

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The Endoplasmic Reticulum (ER)-resident HSP70 chaperone BiP (HSPA5) plays a crucial role in maintaining and restoring protein folding homeostasis in the ER. BiP’s function is often dysregulated in cancer and virus-infected cells, conferring pro-oncogenic and pro-viral advantages. We explored BiP’s functions during infection by the Kaposi’s sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus associated with cancers of immunocompromised patients. Our findings reveal that BiP protein levels are upregulated in infected epithelial cells during the lytic phase of KSHV infection. This upregulation occurs independently of the unfolded protein response (UPR), a major signaling pathway that regulates BiP availability. Genetic and pharmacological inhibition of BiP halts KSHV viral replication and reduces the proliferation and survival of KSHV-infected cells. Notably, inhibition of BiP limits the spread of other alpha- and beta-herpesviruses and poxviruses with minimal toxicity for normal cells. Our work suggests that BiP is a potential target for developing broad-spectrum antiviral therapies against double-stranded DNA viruses and a promising candidate for therapeutic intervention in KSHV-related malignancies.

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“…Notably, by recruiting repressive chromatin regulators to DNA, CRISPRi can repress gene expression not only through proximal promoter elements but also via more distal, enhancer elements. While this tool has been applied widely on the host, it can also effectively repress transcription from the KSHV genome [28], and thus has the potential to provide deep insight into the components and structure of viral transcriptional networks.…”

Section: Introductionmentioning

confidence: 99%

From enhancers to genome conformation: complex transcriptional control underlies expression of a single herpesviral gene

Morgens

Gulyas

Souza

et al. 2023

Preprint

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Gene regulation in eukaryotes relies on many mechanisms for optimal expression, including both protein transcription factors and DNA regulatory elements. CRISPR-based screens of both protein coding genes and non-coding regions have allowed identification of these transcriptional networks in human cells. Double-stranded DNA viruses also invoke human-like regulation to control transcription of viral genes that are required at different stages of the viral lifecycle. Here, we applied CRISPR-based tools to dissect regulation of a viral gene at high resolution in the oncogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), whose compact, densely encoded genome provides unique challenges and opportunities for studying transcriptional networks. Through a combination of CRISPR-interference (CRISPRi) and Cas9 nuclease screening, we mapped a novel regulatory network comprised of coding and noncoding elements that influence expression of the essential KSHV protein ORF68 at early and late stages of the viral lifecycle. ORF68 encodes an essential protein involved in packaging the replicated viral DNA into nascent capsids. Although ORF68 expression initiates early in the viral lifecycle, we found that it is primarily required at later times. This work demonstrates the ability to exhaustively identify features controlling a given locus, essentially capturing a complete viral regulatory circuit that functions within the human nucleus to control transcription.

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CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi’s Sarcoma-Associated Herpesvirus-Infected Cells

Cited by 8 publications

References 52 publications

Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

BiP/GRP78 is a pro-viral factor for diverse dsDNA viruses that promotes the survival and proliferation of cells upon KSHV infection

From enhancers to genome conformation: complex transcriptional control underlies expression of a single herpesviral gene

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