The Restriction of Zoonotic PERV Transmission by Human APOBEC3G

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-CC-3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

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Absence of Replication-Competent Human-Tropic Porcine Endogenous Retroviruses in the Germ Line DNA of Inbred Miniature Swine

Journal of Virology ◽

10.1128/jvi.78.5.2502-2509.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2502-2509 ◽

Cited By ~ 46

Author(s):

Linda Scobie ◽

Samantha Taylor ◽

James C. Wood ◽

Kristen M. Suling ◽

Gary Quinn ◽

...

Keyword(s):

Genomic Dna ◽

Germ Line ◽

Human Cells ◽

Endogenous Retroviruses ◽

Open Reading Frames ◽

Miniature Swine ◽

Dna Libraries ◽

Porcine Endogenous Retroviruses ◽

Perv Transmission

ABSTRACT The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol. 76:3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long env open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.

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Infection of Nonhuman Primate Cells by Pig Endogenous Retrovirus

Journal of Virology ◽

10.1128/jvi.74.16.7687-7690.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7687-7690 ◽

Cited By ~ 56

Author(s):

Juergen H. Blusch ◽

Clive Patience ◽

Yasuhiro Takeuchi ◽

Christian Templin ◽

Christian Roos ◽

...

Keyword(s):

Risk Assessment ◽

Cell Lines ◽

Nonhuman Primate ◽

Endogenous Retrovirus ◽

Papio Hamadryas ◽

Endogenous Retroviruses ◽

Human Donor ◽

Baboon Model ◽

Primate Cell ◽

Perv Transmission

ABSTRACT The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.

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Deaminase-Independent Mode of Antiretroviral Action in Human and Mouse APOBEC3 Proteins

Microorganisms ◽

10.3390/microorganisms8121976 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1976

Author(s):

Yoshiyuki Hakata ◽

Masaaki Miyazawa

Keyword(s):

Molecular Mechanisms ◽

Restriction Factors ◽

Mrna Editing ◽

Antiviral Function ◽

Apobec3 Proteins ◽

And Function ◽

Editing Enzyme ◽

Dependent Pathway ◽

Independent Mode ◽

Human Apobec3g

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) proteins (APOBEC3s) are deaminases that convert cytosines to uracils predominantly on a single-stranded DNA, and function as intrinsic restriction factors in the innate immune system to suppress replication of viruses (including retroviruses) and movement of retrotransposons. Enzymatic activity is supposed to be essential for the APOBEC3 antiviral function. However, it is not the only way that APOBEC3s exert their biological function. Since the discovery of human APOBEC3G as a restriction factor for HIV-1, the deaminase-independent mode of action has been observed. At present, it is apparent that both the deaminase-dependent and -independent pathways are tightly involved not only in combating viruses but also in human tumorigenesis. Although the deaminase-dependent pathway has been extensively characterized so far, understanding of the deaminase-independent pathway remains immature. Here, we review existing knowledge regarding the deaminase-independent antiretroviral functions of APOBEC3s and their molecular mechanisms. We also discuss the possible unidentified molecular mechanism for the deaminase-independent antiretroviral function mediated by mouse APOBEC3.

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Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions

BMC Biology ◽

10.1186/s12915-020-00849-6 ◽

2020 ◽

Vol 18 (1) ◽

Cited By ~ 1

Author(s):

Zhiquan Liu ◽

Siyu Chen ◽

Huanhuan Shan ◽

Yingqi Jia ◽

Mao Chen ◽

...

Keyword(s):

Context Specificity ◽

Base Editing ◽

Human Apobec3g

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Human APOBEC3G Can Restrict Retroviral Infection in Avian Cells and Acts Independently of both UNG and SMUG1

Journal of Virology ◽

10.1128/jvi.02469-07 ◽

2008 ◽

Vol 82 (9) ◽

pp. 4660-4664 ◽

Cited By ~ 40

Author(s):

Marc-André Langlois ◽

Michael S. Neuberger

Keyword(s):

Mammalian Evolution ◽

Retroviral Infection ◽

Cytidine Deaminases ◽

Apobec3 Proteins ◽

Cytidine Deamination ◽

Human Apobec3g ◽

In Cells

ABSTRACT APOBEC3 proteins are mammal-specific cytidine deaminases that can restrict retroviral infection. The exact mechanism of the restriction remains unresolved, but one model envisions that uracilated retroviral cDNA, generated by cytidine deamination, is the target of cellular glycosylases. While restriction is unaffected by UNG deficiency, it has been suggested that the SMUG1 glycosylase might provide a backup. We found that retroviral restriction can be achieved by introducing human APOBEC3G into chicken cells (consistent with the components necessary for APOBEC3-mediated restriction predating mammalian evolution) and used this assay to show that APOBEC3G-mediated restriction can occur in cells deficient in both UNG and SMUG1.

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Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

Nature Chemistry ◽

10.1038/nchem.1795 ◽

2013 ◽

Vol 6 (1) ◽

pp. 28-33 ◽

Cited By ~ 56

Author(s):

Kathy R. Chaurasiya ◽

Micah J. McCauley ◽

Wei Wang ◽

Dominic F. Qualley ◽

Tiyun Wu ◽

...

Keyword(s):

Nucleic Acid ◽

Binding Protein ◽

Nucleic Acid Binding ◽

Nucleic Acid Binding Protein ◽

Acid Binding Protein ◽

Human Apobec3g

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Human APOBEC3G Prevents Emergence of Infectious Endogenous Retrovirus in Mice

Journal of Virology ◽

10.1128/jvi.00728-19 ◽

2019 ◽

Vol 93 (20) ◽

Cited By ~ 6

Author(s):

Rebecca S. Treger ◽

Maria Tokuyama ◽

Huiping Dong ◽

Karen Salas-Briceno ◽

Susan R. Ross ◽

...

Keyword(s):

Ectopic Expression ◽

Endogenous Retrovirus ◽

Endogenous Retroviruses ◽

Toll Like Receptor ◽

Restriction Factors ◽

Cytidine Deaminases ◽

Endogenous Loci ◽

Human Apobec3g

ABSTRACT Endogenous retroviruses (ERV) are found throughout vertebrate genomes, and failure to silence their activation can have deleterious consequences on the host. Mutation and subsequent disruption of ERV loci is therefore an indispensable component of the cell-intrinsic defenses that maintain the integrity of the host genome. Abundant in vitro and in silico evidence have revealed that APOBEC3 cytidine-deaminases, including human APOBEC3G (hA3G), can potently restrict retrotransposition; yet, in vivo data demonstrating such activity is lacking, since no replication-competent human ERV have been identified. In mice deficient for Toll-like receptor 7 (TLR7), transcribed ERV loci can recombine and generate infectious ERV. In this study, we show that ectopic expression of hA3G can prevent the emergence of replication-competent, infectious ERV in Tlr7−/− mice. Mice encode one copy of Apobec3 in their genome. ERV reactivation in Tlr7−/− mice was comparable in the presence or absence of Apobec3. In contrast, expression of a human APOBEC3G transgene abrogated emergence of infectious ERV in the Tlr7−/− background. No ERV RNA was detected in the plasma of hA3G+ Apobec3−/− Tlr7−/− mice, and infectious ERV virions could not be amplified through coculture with permissive cells. These data reveal that hA3G can potently restrict active ERV in vivo and suggest that expansion of the APOBEC3 locus in primates may have helped to provide for the continued restraint of ERV in the human genome. IMPORTANCE Although APOBEC3 proteins are known to be important antiviral restriction factors in both mice and humans, their roles in the restriction of endogenous retroviruses (ERV) have been limited to in vitro studies. Here, we report that human APOBEC3G expressed as a transgene in mice prevents the emergence of infectious ERV from endogenous loci. This study reveals that APOBEC3G can powerfully restrict active retrotransposons in vivo and demonstrates how transgenic mice can be used to investigate host mechanisms that inhibit retrotransposons and reinforce genomic integrity.

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The DNA Deaminase Activity of Human APOBEC3G Is Required for Ty1, MusD, and Human Immunodeficiency Virus Type 1 Restriction

Journal of Virology ◽

10.1128/jvi.02391-07 ◽

2008 ◽

Vol 82 (6) ◽

pp. 2652-2660 ◽

Cited By ~ 128

Author(s):

April J. Schumacher ◽

Guylaine Haché ◽

Donna A. MacDuff ◽

William L. Brown ◽

Reuben S. Harris

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Virus Infectivity ◽

Repair System ◽

Cytosine Deamination ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Human Apobec3g

ABSTRACT Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(ΔVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(ΔVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(ΔVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.

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Expression of HIV-1 Vif and Human APOBEC3G with a Bicistronic Vector

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.108.141 ◽

2011 ◽

Vol 108 ◽

pp. 141-145

Author(s):

Yi Shu Yang ◽

Xiao Na Sun ◽

Di Liu ◽

Xiao Li Wang ◽

Si Si Shen ◽

...

Keyword(s):

Antiviral Activity ◽

Bicistronic Vector ◽

Fluorescent Microscope ◽

Novel Targets ◽

Antiviral Mechanism ◽

Hiv 1 ◽

Human Apobec3g

Novel targets against HIV-1 are booming recently. APOBEC3G has the potential to inhibit the replication of HIV-1, while its antiviral activity is counteracted by Vif. The antiviral mechanism of APOBEC3G and the antagonistic mechanism of Vif have drawn great attention. Considering the occurrence of interaction between Vif and APOBEC3G should be based on that these two proteins coexist in the same cells, a eukaryotic bicistronic vector was adopted to express both Vif and APOBEC3G simultaneously. The expression and distribution of Vif and APOBEC3G proteins are detected with fluorescent microscope.

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