Peter Gee

Host Factor SAMHD1 Restricts DNA Viruses in Non- Dividing Myeloid Cells

SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells. Since DNA viruses utilize cellular dNTPs, we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells. Indeed, two double stranded DNA viruses, vaccinia and herpes simplex virus type 1, are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner. Using a thymidine kinase deficient strain of vaccinia virus, we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1. Therefore, this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses, as well as retroviruses, which infect non-dividing myeloid cells.

Identification of Amino Acids in the Human Tetherin Transmembrane Domain Responsible for HIV-1 Vpu Interaction and Susceptibility

ABSTRACT Tetherin, also known as BST-2/CD317/HM1.24, is an antiviral cellular protein that inhibits the release of HIV-1 particles from infected cells. HIV-1 viral protein U (Vpu) is a specific antagonist of human tetherin that might contribute to the high virulence of HIV-1. In this study, we show that three amino acid residues (I34, L37, and L41) in the transmembrane (TM) domain of human tetherin are critical for the interaction with Vpu by using a live cell-based assay. We also found that the conservation of an additional amino acid at position 45 and two residues downstream of position 22, which are absent from monkey tetherins, are required for the antagonism by Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that an alignment of these four amino acid residues (I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for the Vpu-mediated antagonism of human tetherin. These results contribute to the molecular understanding of human tetherin-specific antagonism by HIV-1 Vpu.

Antimicrobial Peptide LL-37 Produced by HSV-2-Infected Keratinocytes Enhances HIV Infection of Langerhans Cells

Herpes simplex virus (HSV)-2 shedding is associated with increased risk for sexually acquiring HIV. Because Langerhans cells (LCs), the mucosal epithelium resident dendritic cells, are suspected to be one of the initial target cell types infected by HIV following sexual exposure, we examined whether and how HSV-2 affects HIV infection of LCs. Although relatively few HSV-2/HIV-coinfected LCs were detected, HSV-2 dramatically enhanced the HIV susceptibility of LCs within skin explants. HSV-2 stimulated epithelial cell production of antimicrobial peptides (AMPs), including human β defensins and LL-37. LL-37 strongly upregulated the expression of HIV receptors in monocyte-derived LCs (mLCs), thereby enhancing their HIV susceptibility. Culture supernatants of epithelial cells infected with HSV-2 enhanced HIV susceptibility in mLCs, and this effect was abrogated by blocking LL-37 production. These data suggest that HSV-2 enhances sexual transmission of HIV by increasing HIV susceptibility of LCs via epithelial cell production of LL-37.

Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells.

APOBEC1-mediated editing and attenuation of herpes simplex virus 1 DNA indicate that neurons have an antiviral role during herpes simplex encephalitis

ABSTRACT APOBEC1 (A1) is a cytidine deaminase involved in the regulation of lipids in the small intestine. Herpes simplex virus 1 (HSV-1) is a ubiquitous pathogen that is capable of infecting neurons in the brain, causing encephalitis. Here, we show that A1 is induced during encephalitis in neurons of rats infected with HSV-1. In cells stably expressing A1, HSV-1 infection resulted in significantly reduced virus replication compared to that in control cells. Infectivity could be restored to levels comparable to those observed for control cells if A1 expression was silenced by specific A1 short hairpin RNAs (shRNA). Moreover, cytidine deaminase activity appeared to be essential for this inhibition and led to an impaired accumulation of viral mRNA transcripts and DNA copy numbers. The sequencing of viral gene UL54 DNA, extracted from infected A1-expressing cells, revealed G-to-A and C-to-T transitions, indicating that A1 associates with HSV-1 DNA. Taken together, our results demonstrate a model in which A1 induction during encephalitis in neurons may aid in thwarting HSV-1 infection.

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9.

Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy

In the past decade, the development of two innovative technologies, namely, induced pluripotent stem cells (iPSCs) and the CRISPR Cas9 system, has enabled researchers to model diseases derived from patient cells and precisely edit DNA sequences of interest, respectively. In particular, Duchenne muscular dystrophy (DMD) has been an exemplary monogenic disease model for combining these technologies to demonstrate that genome editing can correct genetic mutations in DMD patient-derived iPSCs. DMD is an X-linked genetic disorder caused by mutations that disrupt the open reading frame of the dystrophin gene, which plays a critical role in stabilizing muscle cells during contraction and relaxation. The CRISPR Cas9 system has been shown to be capable of targeting the dystrophin gene and rescuing its expression in in vitro patient-derived iPSCs and in vivo DMD mouse models. In this review, we highlight recent advances made using the CRISPR Cas9 system to correct genetic mutations and discuss how emerging CRISPR technologies and iPSCs in a combined platform can play a role in bringing a therapy for DMD closer to the clinic.

Vpu and BST2: Still Not There Yet?

Extensive investigations have identified two cellular proteins in humans that potently inhibit HIV type 1 (HIV-1) replication and are widely accepted as “restriction factors.” APOBEC3G was identified as a restriction factor that diminishes HIV-1 replication by inducing G-to-A hypermutation in the viral genome, while BST2 has been identified as another restriction factor that impairs the release of nascent virions by tethering them on the surface of infected cells. To counter these restriction factors, HIV-1 has equipped itself with its own weapons: viral infectivity factor (Vif) degrades APOBEC3G, while viral protein U (Vpu) antagonizes BST2. These findings have allowed us to further our understanding of virus–host interaction, namely, the interplay between viral factors versus host restriction factors. In the first case, the interplay between APOBEC3G and Vif is clear: vif-deficient HIV-1 is incapable of replicating in APOBEC3G-expressing cells. This insight directly indicates that APOBEC3G is a bona fide restriction factor and has intrinsic immunity against HIV-1, and that Vif is a prerequisite for HIV-1 infection. In other words, the relationship between Vif and APOBEC3G has already “matured,” and Vif has highly evolved to overcome APOBEC3G. On the other hand, although BST2 drastically impairs the release of vpu-deficient HIV-1 virions, it is puzzling that vpu-deficient HIV-1 is still able to replicate in BST2-expressing cells. These insights imply that BST2-mediated anti-HIV-1 activity is vulnerable, and that Vpu is dispensable for HIV-1 infection. If so, why has Vpu acquired the counteracting potential against BST2? Was it necessary or important for HIV-1? Or is the relationship between Vpu and BST2 still “immature”? In this review, we particularly focus on the interplay between Vpu and BST2. We discuss the possibility that Vpu has evolved as a potent antagonist against BST2, and finally, propose a hypothesis that Vpu has evolved as a promoter of human-to-human HIV-1 transmission. Since the first report of acquired immunodeficiency syndrome patients in 1981, HIV-1 has spread explosively worldwide and is currently a pandemic. This review proposes a concept suggesting that the current HIV-1 pandemic may be partly attributed by Vpu.

Perspectives of Genome-Editing Technologies for HIV Therapy.

Background: Current HIV antiretroviral therapies potently suppress virus replication and prevent patients from progressing to AIDS but are unable to completely eliminate HIV due to the existence of dormant viral reservoirs which threaten to reemerge at anytime. Recently, genome-editing technologies that can recognize specific DNA sequences, including viral DNA, are being touted as promising tools for curing HIV, owing to their specificity, ease of use, and ability to be custom designed. Conclusion: Here, we introduce several novel strategies aimed at eradicating HIV proviruses with state-of-the-art genome-editing technologies and discuss perspectives of these approaches for curing HIV.

Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells

Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. Thus, CRONUS provides a novel platform for modeling diseases and genetic variations.