Restriction of retroviral replication by APOBEC3G/F and TRIM5alpha

Pathogenic viral infections have exerted selection pressure on their hosts to evolve cellular antiviral inhibitors referred to as restriction factors. Examples of such molecules are APOBEC3G, APOBEC3F and TRIM5alpha. APOBEC3G and APOBEC3F are cytidine deaminases that are able to strongly inhibit retroviral replication by at least two mechanisms. They are counteracted by the lentiviral Vif protein. TRIM5alpha binds to sensitive, incoming retroviruses via its C-terminal PRY/SPRY domain and rapidly recruits them to the proteasome before significant viral DNA synthesis can occur. Both of these proteins robustly block retroviral replication in a species-specific way. It remains an open but important question as to whether innate restriction factors such as these can be harnessed to inhibit HIV-1 replication in humans.     

DNA-uracil and human pathology

Uracil is usually an inappropriate base in DNA, but it is also a normal intermediate during somatic hypermutation (SHM) and class switch recombination (CSR) in adaptive immunity. In addition, uracil is introduced into retroviral DNA by the host as part of a defence mechanism. The sources of uracil in DNA are spontaneous or enzymatic deamination of cytosine (U:G mispairs) and incorporation of dUTP (U:A pairs). Uracil in DNA is removed by a uracil-DNA glycosylase. The major ones are nuclear UNG2 and mitochondrial UNG1 encoded by the UNG-gene, and SMUG1 that also removes oxidized pyrimidines, e.g. 5-hydroxymethyluracil. The other ones are TDG that removes U and T from mismatches, and MBD4 that removes U from CpG contexts. UNG2 is found in replication foci during the S-phase and has a distinct role in repair of U:A pairs, but it is also important in U:G repair, a function shared with SMUG1. SHM is initiated by activation-induced cytosine deaminase (AID), followed by removal of U by UNG2. Humans lacking UNG2 suffer from recurrent infections and lymphoid hyperplasia, and have skewed SHM and defective CSR, resulting in elevated IgM and strongly reduced IgG, IgA and IgE. UNG-defective mice also develop B-cell lymphoma late in life. In the defence against retrovirus, e.g. HIV-1, high concentrations of dUTP in the target cells promotes misincorporation of dUMP-, and host cell APOBEC proteins may promote deamination of cytosine in the viral DNA. This facilitates degradation of viral DNA by UNG2 and AP-endonuclease. However, viral proteins Vif and Vpr counteract this defense by mechanisms that are now being revealed. In conclusion, uracil in DNA is both a mutagenic burden and a tool to modify DNA for diversity or degradation.     

Uracil-DNA glycosylase in base excision repair and adaptive immunity: species differences between man and mouse

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung(+/+) and Ung(-/-) backcrossed mice. Interestingly, human cells displayed ～15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ～8-fold higher in mouse cells, constituting ～50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung(-/-) mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity.     

Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms

DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.     

Adaptive evolution and antiviral activity of the conserved mammalian cytidine deaminase APOBEC3H

The APOBEC3 genes encode cytidine deaminases that act as components of an intrinsic immune defense that have potent activity against a variety of retroelements. This family of genes has undergone a rapid expansion from one or two genes in nonprimate mammals to at least seven members in primates. Here we describe the evolution and function of an uncharacterized antiviral effector, APOBEC3H, which represents the most evolutionarily divergent APOBEC3 gene found in primates. We found that APOBEC3H has undergone significant adaptive evolution in primates. Consistent with our previous findings implicating adaptively evolving APOBEC3 genes as antiviral effectors, APOBEC3H from Old World monkeys (OWMs) has efficient antiviral activity against primate lentiviruses, is sensitive to inactivation by the simian immunodeficiency virus Vif protein, and is capable of hypermutating retroviral genomes. In contrast, human APOBEC3H is inherently poorly expressed in primate cells and is ineffective at inhibiting retroviral replication. Both OWM and human APOBEC3H proteins can be expressed in bacteria, where they display significant DNA mutator activity. Thus, humans have retained an APOBEC3H gene that encodes a functional, but poorly expressed, cytidine deaminase with no apparent antiviral activity. The consequences of the lack of antiviral activity of human APOBEC3H are likely to be relevant to the current-day abilities of humans to combat retroviral challenges.     

SMUG1 is able to excise uracil from immunoglobulin genes: insight into mutation versus repair

Mammals harbour multiple enzymes capable of excising uracil from DNA, although their distinct physiological roles remain uncertain. One of them (UNG) plays a critical role in antibody gene diversification, as UNG deficiency alone is sufficient to perturb the process. Here, we show this unique requirement for UNG does not reflect the fact that other glycosylases are unable to access the U:G lesion. SMUG1, if overexpressed, can partially substitute for UNG to assist antibody diversification as judged by its effect on somatic hypermutation patterns (in both DT40 B cells and mice) as well as a restoration of isotype switching in SMUG-transgenic msh2-/- ung-/- mice. However, SMUG1 plays little natural role in antibody diversification because (i) it is diminishingly expressed during B-cell activation and (ii) even if overexpressed, SMUG1 more appears to favour conventional repair of the uracil lesion than assist diversification. The distinction between UNG and overexpressed SMUG1 regarding the balance between antibody diversification and non-mutagenic repair of the U:G lesion could reflect the association of UNG (but not SMUG1) with sites of DNA replication.     

Strikingly different properties of uracil-DNA glycosylases UNG2 and SMUG1 may explain divergent roles in processing of genomic uracil

Genomic uracil resulting from spontaneously deaminated cytosine generates mutagenic U:G mismatches that are usually corrected by error-free base excision repair (BER). However, in B-cells, activation-induced cytosine deaminase (AID) generates U:G mismatches in hot-spot sequences at Ig loci. These are subject to mutagenic processing during somatic hypermutation (SHM) and class switch recombination (CSR). Uracil N-glycosylases UNG2 and SMUG1 (single strand-selective monofunctional uracil-DNA glycosylase 1) initiate error-free BER in most DNA contexts, but UNG2 is also involved in mutagenic processing of AID-induced uracil during the antibody diversification process, the regulation of which is not understood. AID is strictly single strand-specific. Here we show that in the presence of Mg2+ and monovalent salts, human and mouse SMUG1 are essentially double strand-specific, whereas UNG2 efficiently removes uracil from both single and double stranded DNA under all tested conditions. Furthermore, SMUG1 and UNG2 display widely different sequence preferences. Interestingly, uracil in a hot-spot sequence for AID is 200-fold more efficiently removed from single stranded DNA by UNG2 than by SMUG1. This may explain why SMUG1, which is not excluded from Ig loci, is unable to replace UNG2 in antibody diversification. We suggest a model for mutagenic processing in which replication protein A (RPA) recruits UNG2 to sites of deamination and keeps DNA in a single stranded conformation, thus avoiding error-free BER of the deaminated cytosine.     

Structure and specificity of the vertebrate anti-mutator uracil-DNA glycosylase SMUG1

Cytosine deamination is a major promutagenic process, generating G:U mismatches that can cause transition mutations if not repaired. Uracil is also introduced into DNA via nonmutagenic incorporation of dUTP during replication. In bacteria, uracil is excised by uracil-DNA glycosylases (UDG) related to E. coli UNG, and UNG homologs are found in mammals and viruses. Ung knockout mice display no increase in mutation frequency due to a second UDG activity, SMUG1, which is specialized for antimutational uracil excision in mammalian cells. Remarkably, SMUG1 also excises the oxidation-damage product 5-hydroxymethyluracil (HmU), but like UNG is inactive against thymine (5-methyluracil), a chemical substructure of HmU. We have solved the crystal structure of SMUG1 complexed with DNA and base-excision products. This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU.     

Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities

Human APOBEC3F and APOBEC3G are double-domained deaminases that can catalyze dC→dU deamination in HIV-1 and MLV retroviral DNA replication intermediates, targeting T–C or C–C dinucleotides, respectively. HIV-1 antagonizes their action through its vif gene product, which has been shown (at least in the case of APOBEC3G) to interact with the N-terminal domain of the deaminase, triggering its degradation. Here, we compare APOBEC3F and APOBEC3G to APOBEC3C, a single-domained deaminase that can also act on both HIV-1 and MLV. We find that whereas APOBEC3C contains all the information necessary for both Vif-binding and cytidine deaminase activity in a single domain, it is the C-terminal domain of APOBEC3F and APOBEC3G that confer their target site specificity for cytidine deamination. We have exploited the fact that APOBEC3C, whilst highly homologous to the C-terminal domain of APOBEC3F, exhibits a distinct target site specificity (preferring Y–C dinucleotides) in order to identify residues in APOBEC3F that might affect its target site specificity. We find that this specificity can be altered by single amino acid substitutions at several distinct positions, suggesting that the strong dependence of APOBEC3-mediated deoxycytidine deamination on the 5′-flanking nucleotide is sensitive to relatively subtle changes in the APOBEC3 structure. The approach has allowed the isolation of APOBEC3 DNA mutators that exhibit novel target site preferences.     

Evidence for Restriction of Ancient Primate Gammaretroviruses by APOBEC3 but Not TRIM5α Proteins

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5α and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a ‘fossil record’ of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5α proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species–dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.     

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30-40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained approximately 1% of the viral genomic RNA but approximately 30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.     

The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover

Background

The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.

Methodology/Principal Findings

We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.

Conclusions/Significance

This work identifies UNG2 and SMUG1 as novel targets for CRL4^DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4^DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4^DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.     

Selective inhibition of Alu retrotransposition by APOBEC3G

The non-LTR retrotransposon LINE-1 (L1) comprises  17% of the human genome, and the L1-encoded proteins can function in trans to mediate the retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other L1-mediated retrotransposition processes. We demonstrate that APOBEC3G selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An active cytidine deaminase site is not required for the inhibition of Alu retrotransposition and the resultant integration events lack G to A or C to T hypermutation. These data demonstrate a differential restriction of L1 and Alu retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein complex may be targeted by APOBEC3G.     

Evidence for restriction of ancient primate gammaretroviruses by APOBEC3 but not TRIM5alpha proteins

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5alpha and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a 'fossil record' of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5alpha proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species-dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.     

Repression of porcine endogenous retrovirus infection by human APOBEC3 proteins

It has been shown that porcine endogenous retrovirus (PERV) can infect human cells, indicating that PERV transmission poses a serious concern in pig-to-human xenotransplantation. A number of recent studies have reported on retrovirus interference by antiviral proteins. The most potent antiviral proteins are members of the APOBEC family of cytidine deaminases, which are involved in defense against retroviral attack. These proteins are present in the cytoplasm of mammalian cells and inhibit retroviral replication. To evaluate the inhibition of PERV transmission by human APOBEC3 proteins, we co-transfected 293T cells with a PERV molecular clone and human APOBEC3F or APOBEC3G expression vectors, and monitored PERV replication competency using a quantitative analysis of PERV pol genes. The replication of PERVs in cells co-expressing human APOBEC3s was reduced by 60–90% compared with PERV-only control. These results suggest that human APOBEC3G and APOBEC3F might serve a potential barrier function against PERV transmission in xenotransplantation.