Kate N. Bishop

DNA deamination mediates innate immunity to (retro)viral infection

CEM15/APOBEC3G is a cellular protein required for resistance to infection by virion infectivity factor (Vif)-deficient human immunodeficiency virus (HIV). Here, using a murine leukemia virus (MLV)-based system, we provide evidence that CEM15/APOBEC3G is a DNA deaminase that is incorporated into virions during viral production and subsequently triggers massive deamination of deoxycytidine to deoxyuridine within the retroviral minus (first)-strand cDNA, thus providing a probable trigger for viral destruction. Furthermore, HIV Vif can protect MLV from this CEM15/APOBEC3G-dependent restriction. These findings imply that targeted DNA deamination is a major strategy of innate immunity to retroviruses and likely also contributes to the sequence variation observed in many viruses (including HIV).

Cytidine Deamination of Retroviral DNA by Diverse APOBEC Proteins

The human cytidine deaminase APOBEC3G edits both nascent human immunodeficiency virus (HIV) and murine leukemia virus (MLV) reverse transcripts, resulting in loss of infectivity. The HIV Vif protein is able to protect both viruses from this innate restriction to infection. Here, we demonstrate that a number of other APOBEC family members from both humans and rodents can mediate anti-HIV effects, through cytidine deamination. Three of these, rat APOBEC1, mouse APOBEC3, and human APOBEC3B, are able to inhibit HIV infectivity even in the presence of Vif. Like APOBEC3G, human APOBEC3F preferentially restricts vif-deficient virus. Indeed, the mutation spectra and expression profile found for APOBEC3F indicate that this enzyme, together with APOBEC3G, accounts for the G to A hypermutation of proviruses described in HIV-infected individuals. Surprisingly, although MLV infectivity is acutely reduced by APOBEC3G, no other family member tested here had this effect. It is especially interesting that although both rodent APOBECs markedly diminish wild-type HIV infectivity, MLV is resistant to these proteins. This implies that MLV may have evolved to avoid deamination by mouse APOBEC3. Overall, our findings show that although APOBEC family members are highly related, they exhibit significantly distinct antiviral characteristics that may provide new insights into host-pathogen interactions.

Antiviral Potency of APOBEC Proteins Does Not Correlate with Cytidine Deamination

ABSTRACT The human cytidine deaminases APOBEC3G (hA3G) and APOBEC3F (hA3F) are intracellular antiretroviral factors that can hypermutate nascent reverse transcripts and inhibit the replication of human immunodeficiency virus type 1 (HIV-1). Both enzymes have two cytidine deaminase motifs, although only the C-terminal motif is catalytic. Current models of APOBEC protein function imply editing is the principal mechanism of antiviral activity. In particular, hA3G is a more potent inhibitor of HIV-1 infectivity than hA3F and also induces a greater frequency of mutations in HIV-1 cDNA. We used hA3G/hA3F chimeric proteins to investigate whether cytidine deaminase potential reflects antiviral potency. We show here that the origin of the C-terminal deaminase motif is sufficient to determine the degree of mutation induced in a bacterial assay that measures mutations in chromosomal DNA. In contrast, this was not the case in the context of HIV-1 infection where the N-terminal deaminase motif also modulated the editing capabilities of the chimeras. Surprisingly, although three of the chimeric proteins induced levels of mutation that approximated those of parental hA3F, they displayed lower levels of antiviral activity. Most importantly, real-time PCR experiments revealed that the quantity of reverse transcripts detected in target cells, rather than the mutational burden carried by such DNAs, corresponded closely with viral infectivity. In other words, the antiviral phenotype of APOBEC proteins correlates with their ability to prevent the accumulation of reverse transcripts and not with the induction of hypermutation.

APOBEC3G inhibits elongation of HIV-1 reverse transcripts

APOBEC3G (A3G) is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

APOBEC3F Can Inhibit the Accumulation of HIV-1 Reverse Transcription Products in the Absence of Hypermutation COMPARISONS WITH APOBEC3G

APOBEC3F (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like protein 3F) is a cytidine deaminase that, like APOBEC3G, is able to restrict the replication of HIV-1/ Δvif. Initial studies revealed high numbers of mutations in the cDNA of viruses produced in the presence of these proteins, suggesting that cytidine deamination underpinned the inhibition of infection. However, we have recently shown that catalytically inactive APOBEC3G proteins, derived through mutation of the C-terminal cytidine deaminase motif, still exert a substantial antiviral effect. Here, we have generated a panel of APOBEC3F mutant proteins and show that the C-terminal cytidine deaminase motif is essential for catalytic activity and that catalytic activity is not necessary for the antiviral effect of APOBEC3F. Furthermore, we demonstrate that the antiviral activities of wild-type and catalytically inactive APOBEC3F and APOBEC3G proteins correspond well with reductions in the accumulation of viral reverse transcription products. Additional comparisons between APOBEC3F and APOBEC3G suggest that the loss of deaminase activity is more detrimental to APOBEC3G function than to APOBEC3F function, as reflected by perturbations to the suppression of reverse transcript accumulation as well as antiviral activity. Taken together, these data suggest that both APOBEC3F and APOBEC3G are able to function as antiviral factors in the absence of cytidine deamination, that this editing-independent activity is an important aspect of APOBEC protein-mediated antiviral phenotypes, but that APOBEC3F may be a better model in which to study it.

Use of a Transient Assay for Studying the Genetic Determinants of Fv1 Restriction

ABSTRACT To probe the genetic determinants controlling the interaction between the retroviral restriction gene Fv1 and its murine leukemia virus target, we set out to develop rapid, transient assays for Fv1 function. Cells were transfected or transduced withFv1 expression plasmids which can produce green fluorescent protein via an internal ribosome entry site positioned between theFv1 and green fluorescent protein coding sequences.Fv1 function was then assessed by comparing virus replication in green fluorescent protein-positive and -negative cells, using retroviral vectors encoding a second fluorescent marker, yellow fluorescent protein, or β-galactosidase. Using this assay, we could show that Fv1 specificities were not as absolute as previously thought, since the Fv1b allele was capable of interacting with “nonrestricted” B- and NB-tropic viruses and by shuffling the n- and b-alleles of Fv1, it was possible to generate a Fv1 molecule capable of restricting N-, B-, and NB-tropic viruses equally efficiently. Further, we could show that the presence of nonrestricting Fv1 in the same cell as restrictive Fv1 abrogates restriction, implying competition for binding to the retroviral target.

Susceptibility of xenotropic murine leukemia virus-related virus (XMRV) to retroviral restriction factors

Xenotropic murine leukemia virus-related virus (XMRV) is a recently discovered gammaretrovirus that has been linked to prostate cancer and chronic fatigue syndrome. This virus is therefore an important potential human pathogen and, as such, it is essential to understand its host cell tropism. Intriguingly, infectious virus has been recovered from patient-derived peripheral blood mononuclear cells. These cells express several antiviral restriction factors that are capable of inhibiting the replication of a wide range of retroviruses, including other gamma retroviruses. This raises the possibility that, similar to HIV, XMRV may have acquired resistance to restriction. We therefore investigated the susceptibility of XMRV to a panel of different restriction factors. We found that both human APOBEC3 and tetherin proteins are able to block XMRV replication. Expression of human TRIM5α, however, had no effect on viral infectivity. There was no evidence that XMRV expressed countermeasures to overcome restriction. In addition, the virus was inhibited by factors from nonhuman species, including mouse Apobec3, tetherin, and Fv1 proteins. These results have important implications for predicting the natural target cells for XMRV replication, for relating infection to viral pathogenicity and pathology, and for the design of model systems with which to study XMRV-related diseases.

Conserved Footprints of APOBEC3G on Hypermutated Human Immunodeficiency Virus Type 1 and Human Endogenous Retrovirus HERV-K(HML2) Sequences

ABSTRACT The human polynucleotide cytidine deaminases APOBEC3G (hA3G) and APOBEC3F (hA3F) are antiviral restriction factors capable of inducing extensive plus-strand guanine-to-adenine (G-to-A) hypermutation in a variety of retroviruses and retroelements, including human immunodeficiency virus type 1 (HIV-1). They differ in target specificity, favoring plus-strand 5′GG and 5′GA dinucleotide motifs, respectively. To characterize their mutational preferences in detail, we analyzed single-copy, near-full-length HIV-1 proviruses which had been hypermutated in vitro by hA3G or hA3F. hA3-induced G-to-A mutation rates were significantly influenced by the wider sequence context of the target G. Moreover, hA3G, and to a lesser extent hA3F, displayed clear tetranucleotide preference hierarchies, irrespective of the genomic region examined and overall hypermutation rate. We similarly analyzed patient-derived hypermutated HIV-1 genomes using a new method for estimating reference sequences. The majority of these, regardless of subtype, carried signatures of hypermutation that strongly correlated with those induced in vitro by hA3G. Analysis of genome-wide hA3-induced mutational profiles confirmed that hypermutation levels were reduced downstream of the polypurine tracts. Additionally, while hA3G mutations were found throughout the genome, hA3F often intensely mutated shorter regions, the locations of which varied between proviruses. We extended our analysis to human endogenous retroviruses (HERVs) from the HERV-K(HML2) family, finding two elements that carried clear footprints of hA3G activity. This constitutes the most direct evidence to date for hA3G activity in the context of natural HERV infections, demonstrating the involvement of this restriction factor in defense against retroviral attacks over millions of years of human evolution.

Further Investigation of Simian Immunodeficiency Virus Vif Function in Human Cells

ABSTRACT Primate lentivirus Vif proteins function by suppressing the antiviral activity of the cell-encoded apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) proteins APOBEC3G and APOBEC3F. It has been hypothesized that species-specific susceptibilities of APOBEC proteins to Vif proteins may help govern the transmission of primate lentiviruses to new host species. Consistent with this view and with previous results, we report that the Vif proteins of several diverse simian immunodeficiency viruses (SIVs) that are not known to infect humans are not effective inhibitors of human APOBEC3G or APOBEC3F when assessed in transient-transfection experiments. Unexpectedly, this lack of SIV Vif function did not prevent the replication of two vif-deficient SIVs (SIVtan and SIVmnd1; isolated from tantalus monkeys and mandrills, respectively) in a human T-cell line, HUT78, that expresses both APOBEC 3G and APOBEC3F, a finding which demonstrates that some SIVs are partially resistant to the antiretroviral effects of these enzymes irrespective of Vif function. Additional virus replication studies also revealed that the Vif protein of SIVtan is, in fact, active in human T cells, as it substantially enhanced the replication of its cognate virus and human immunodeficiency virus type 1. In sum, we now consider it improbable that species-specific restrictions to SIV Vif function can explain the lack of human infection with certain SIVs. Instead, our data reveal that the species-specific modulation of Vif function is more complex than previously envisioned and that additional (as-yet-unidentified) viral or host factors may be involved in regulating this dynamic interaction between host and pathogen.

Identification of the Regions of Fv1 Necessary for Murine Leukemia Virus Restriction

ABSTRACT The Fv1 gene restricts murine leukemia virus replication via an interaction with the viral capsid protein. To study this interaction, a number of mutations, including a series of N-terminal and C-terminal deletions, internal deletions, and a number of single-amino-acid substitutions, were introduced into the n and b alleles of the Fv1 gene and the effects of these changes on virus restriction were measured. A significant fraction of the Fv1 protein was not required for restriction; however, retention of an intact major homology region as well as of domains toward the N and C termini was essential. Binding specificity appeared to be a combinatorial property of a number of residues within the C-terminal portion of Fv1.