Opossum APOBEC1 is a DNA mutator with retrovirus and retroelement restriction activity.

APOBEC3s (A3s) are single-stranded DNA cytosine deaminases that provide innate immune defences against retroviruses and mobile elements. A3s are specific to eutherian mammals because no direct homologs exist at the syntenic genomic locus in metatherian (marsupial) or prototherian (monotreme) mammals. However, the A3s in these species have the likely evolutionary precursors, the antibody gene deaminase AID and the RNA/DNA editing enzyme APOBEC1 (A1). Here, we used cell culture-based assays to determine whether opossum A1 restricts the infectivity of retroviruses including human immunodeficiency virus type 1 (HIV-1) and the mobility of LTR/non-LTR retrotransposons. Opossum A1 partially inhibited HIV-1, as well as simian immunodeficiency virus (SIV), murine leukemia virus (MLV), and the retrotransposon MusD. The mechanism of inhibition required catalytic activity, except for human LINE1 (L1) restriction, which was deamination-independent. These results indicate that opossum A1 functions as an innate barrier to infection by retroviruses such as HIV-1, and controls LTR/non-LTR retrotransposition in marsupials.

Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities.

APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

GANP interacts with APOBEC3G and facilitates its encapsidation into the virions to reduce HIV-1 infectivity.

The ssDNA-dependent deoxycytidine deaminase apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (A3G) is a potent restrictive factor against HIV-1 virus lacking viral-encoded infectivity factor (Vif) in CD4(+) T cells. A3G antiretroviral activity requires its encapsulation into HIV-1 virions. In this study, we show that germinal center-associated nuclear protein (GANP) is induced in activated CD4(+) T cells and physically interacts with A3G. Overexpression of GANP augments the A3G encapsidation into the virion-like particles and ΔVif HIV-1 virions. GANP is encapsidated in HIV-1 virion and modulates A3G packaging into the cores together with cellular RNAs, including 7SL RNA, and with unspliced HIV-1 genomic RNA. GANP upregulation leads to a significant increase in A3G-catalyzed G→A hypermutation in the viral genome and suppression of HIV-1 infectivity in a single-round viral infection assay. Conversely, GANP knockdown caused a marked increase in HIV-1 infectivity in a multiple-round infection assay. The data suggest that GANP is a cellular factor that facilitates A3G encapsidation into HIV-1 virions to inhibit viral infectivity.

Intrinsic immunity against retrotransposons by APOBEC cytidine deaminases.

Over 40% of the human genome is recognizable as having been derived from ancient retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate, with an additional few percent classified as DNA transposable elements. Endogenous retroviruses are long terminal repeat (LTR)-type retroelements that account for ~8% of human genomic DNA. Non-LTR members are present at extremely high copy numbers, with ~17% of the human genome consisting of long interspersed nuclear elements (LINEs). These LINEs modify vertebrate genomes not only through insertions, but also by the indirect replication of non-autonomous retrotransposons, such as short interspersed nuclear elements. As expected, vertebrate intrinsic immunity has evolved to support a balance between retroelement insertions that confer beneficial genetic diversity and those that cause deleterious gene disruptions. The mammalian cytidine deaminases encoded by the APOBEC3 genes can restrict a broad number of exogenous pathogens, such as exogenous retroviruses, and the mobility of endogenous retroelements. Furthermore, APOBEC1 from a variety of mammalian species, which mediates the cytidine (C) to uridine (U) deamination of apolipoprotein B (apoB) mRNA, a protein involved in lipid transport, also plays a role in controlling mobile elements. These mammalian apoB mRNA-editing, catalytic polypeptide (APOBEC) cytidine deaminases, which can bind to single-stranded DNA (ssDNA) as well as RNA, are able to insert mutations into ssDNA and/or RNA as a result of their ability to deaminate C to U. While these APOBEC cytidine deaminases with DNA mutagenic activity can be deleterious to cells, their biological modifications, such as protein-protein interactions and subcellular localization, in addition to their ability to bind to RNA, appear to have conferred a role for APOBECs as a cellular defense system against retroviruses and retroelements. In support of this notion, the expansion of the single APOBEC3 gene in mice to the seven APOBEC3 genes found in primates apparently correlates with the significant enhancement of the restriction of endogenous retroelements seen in primates, including humans. This review discusses the current understanding of the mechanism of action of APOBEC cytidine deaminases and attempts to summarize their roles in controlling retrotransposons.

Apolipoprotein B mRNA-editing, catalytic polypeptide cytidine deaminases and retroviral restriction.

Apolipoprotein B (apo B) messenger RNA (mRNA)-editing, catalytic polypeptide (APOBEC) cytidine deaminases (CDAs), which can insert mutations into DNA and/or RNA as a result of their ability to deaminate cytidine (C) to uridine (U), originated from a branch of the zinc-dependent deaminase superfamily at the beginning of vertebrate radiation. The ability of mammalian CDAs encoded by the APOBEC3 genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including human immunodeficiency virus-1, is well established. Furthermore, APOBEC1 from a variety of mammalian species, which mediates the C-to-U deamination of apo B mRNA, a protein involved in lipid transport, also has a role in controlling mobile elements. A large portion of the mammalian genome is derived from ancient transposable elements. Retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate, constitute the majority of these mobile genetic elements. Endogenous retroviruses are long-terminal repeat (LTR)-type retroelements that account for approximately 10% of human and murine genomic DNA. Non-LTR members are present in extremely high copy numbers, with approximately 40% of the human and murine genomes consisting of long-interspersed nuclear element-1 (L1). These L1 elements modify mammalian genomes not only through insertions but also by the indirect replication of non-autonomous retrotransposons. As expected, vertebrate intrinsic immunity has evolved to support a balance between retroelement insertions that cause deleterious gene disruptions and those that confer beneficial genetic diversity. This review discusses the current understanding of the mechanism of action of APOBEC CDAs and their role in controlling retroviruses and retroelements.

Vipirinin, a coumarin-based HIV-1 Vpr inhibitor, interacts with a hydrophobic region of VPR.

The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) is an accessory protein that has been shown to have multiple roles in HIV-1 pathogenesis. By screening chemical libraries in the RIKEN Natural Products Depository, we identified a 3-phenyl coumarin-based compound that inhibited the cell cycle arrest activity of Vpr in yeast and Vpr-dependent viral infection of human macrophages. We determined its minimal pharmacophore through a structure-activity relationship study and produced more potent derivatives. We detected direct binding, and by assaying a panel of Vpr mutants, we found the hydrophobic region about residues Glu-25 and Gln-65 to be potentially involved in the binding of the inhibitor. Our findings exposed a targeting site on Vpr and delineated a convenient approach to explore other targeting sites on the protein using small molecule inhibitors as bioprobes.

Intrinsic restriction activity by apolipoprotein B mRNA editing enzyme APOBEC1 against the mobility of autonomous retrotransposons.

The ability of mammalian cytidine deaminases encoded by the APOBEC3 (A3) genes to restrict a broad number of endogenous retroelements and exogenous retroviruses, including murine leukemia virus and human immunodeficiency virus (HIV)-1, is now well established. The RNA editing family member apolipoprotein B (apo B)-editing catalytic subunit 1 (APOBEC1; A1) from a variety of mammalian species, a protein involved in lipid transport and which mediates C-U deamination of mRNA for apo B, has also been shown to modify a range of exogenous retroviruses, but its activity against endogenous retroelements remains unclear. Here, we show in cell culture-based retrotransposition assays that A1 family proteins from multiple mammalian species can also reduce the mobility and infectivity potential of LINE-1 (long interspersed nucleotide sequence-1, L1) and long-terminal repeats (LTRs) retrotransposons (or endogenous retroviruses), such as murine intracisternal A-particle (IAP) and MusD sequences. The anti-L1 activity of A1 was mainly mediated by a deamination-independent mechanism, and was not affected by subcellular localization of the proteins. In contrast, the inhibition of LTR-retrotransposons appeared to require the deaminase activity of A1 proteins. Thus, the AID/APOBEC family proteins including A1s employ multiple mechanisms to regulate the mobility of autonomous retrotransposons in several mammalian species.

Intrinsic restriction activity by AID/APOBEC family of enzymes against the mobility of retroelements.

A large portion of the mammalian genome is derived from ancient transposable elements. Retroelements, transported by an intracellular copy-and-paste process involving an RNA intermediate (retrotransposition), constitute a majority of these mobile genetic elements. Endogenous retroviruses are LTR-type retroelements accounting for around 8% of human or murine genomic DNA. Non-LTR members are present in extremely high copy numbers; with LINE-1 contributing to nearly 40% of human and murine genomes. These LINE-1 elements modify mammalian genomes not only through insertions, but also by indirect replication of nonautonomous retrotransposons such as SINEs. As expected, cellular machineries of vertebrate's innate immunity have evolved to support a balance between retroelement insertions that cause deleterious gene disruptions and those that confer beneficial genetic diversity. The ability of APOBEC3 cytidine deaminases targeting DNA to restrict a broad number of retroviruses and retro-elements is now well established. More recently, the RNA editing family member APOBEC1, a protein involved in lipid transport, has also been shown to be involved in keeping mobile elements under control. This review discusses current understanding of the mechanism of action of the AID/APOBEC family, and their role in controlling the retrotransposition of endogenous retroelements.

APOBEC3G generates nonsense mutations in human T-cell leukemia virus type 1 proviral genomes in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) induces cell proliferation after infection, leading to efficient transmission by cell-to-cell contact. After a long latent period, a fraction of carriers develop adult T-cell leukemia (ATL). Genetic changes in the tax gene in ATL cells were reported in about 10% of ATL cases. To determine genetic changes that may occur throughout the provirus, we determined the entire sequence of the HTLV-1 provirus in 60 ATL cases. Abortive genetic changes, including deletions, insertions, and nonsense mutations, were frequent in all viral genes except the HBZ gene, which is transcribed from the minus strand of the virus. G-to-A base substitutions were the most frequent mutations in ATL cells. The sequence context of G-to-A mutations was in accordance with the preferred target sequence of human APOBEC3G (hA3G). The target sequences of hA3G were less frequent in the plus strand of the HBZ coding region than in other coding regions of the HTLV-1 provirus. Nonsense mutations in viral genes including tax were also observed in proviruses from asymptomatic carriers, indicating that these mutations were generated during reverse transcription and prior to oncogenesis. The fact that hA3G targets the minus strand during reverse transcription explains why the HBZ gene is not susceptible to such nonsense mutations. HTLV-1-infected cells likely take advantage of hA3G to escape from the host immune system by losing expression of viral proteins.

The antiretroviral potency of APOBEC1 deaminase from small animal species.

Although the role of the APOBEC3-dependent retroelement restriction system as an intrinsic immune defense against human immunodeficiency virus type1 (HIV-1) infection is becoming clear, only the rat ortholog of mammalian APOBEC1s (A1) thus far has been shown to possess antiviral activity. Here, we cloned A1 cDNAs from small animal species, and showed that similar to rat A1, both wild-type and Deltavif HIV-1 infection was inhibited by mouse and hamster A1 (4- to 10-fold), whereas human A1 had negligible effects. Moreover, rabbit A1 significantly reduced the infectivity of both HIV-1 virions (>300-fold), as well as that of SIVmac, SIVagm, FIV and murine leukemia virus. Immunoblot analysis showed that A1s were efficiently incorporated into the HIV-1 virion, and their packaging is mediated through an interaction with the nucleocapsid Gag domain. Interestingly, there was a clear accumulation of particular C-T changes in the genomic RNAs of HIV-1 produced in their presence, with few G-A changes in the proviral DNA. Together, these data reveal that A1 may function as a defense mechanism, regulating retroelements in a wide range of mammalian species.

Current topics in prevention of human T-cell leukemia virus type i infection: NF-kappa B inhibitors and APOBEC3.

Human T-cell leukemia virus type I (HTLV-I) is the first human retrovirus and causes adult T-cell leukemia/lymphoma (ATL). Constitutive activation of nuclear factor-kappa B (NF-kappa B) in the leukemic cells is essential for their growth and survival. Thus, NF-kappa B inhibitors have been attracting attention as a potential strategy to treat ATL. Recently, the field of retrovirus research has been stimulated by the discovery of an innate host defense factor, APOBEC3, against the retroviruses. HTLV-I is relatively resistant to the antiviral effects of APOBEC3. To clarify the resistance of HTLV-I against APOBEC3 might contribute to the design of effective therapeutic approaches.

Recurrent HIV-1 integration at the BACH2 locus in resting CD4+ T cell populations during effective highly active antiretroviral therapy.

The persistence of latent human immunodeficiency virus type 1 (HIV-1) has been considered one of the major obstacles for eradication of the virus in infected individuals receiving successful antiretroviral therapy. To determine the contribution of integration sites to viral latency within clinical settings, an inverse polymerase chain reaction method was used to analyze integration sites in CD4(+) T cells from patients showing long-term undetectable plasma viral RNA. Of 457 sites identified in 7 patients, almost all (96%) resided within transcriptional units, usually in introns of the human genome. Studies of 18 genes in which HIV-1 integrates found them to be actively expressed in resting CD4(+) T cells. On the other hand, integration sites in the alpha satellite region was also identified in some patients, albeit at low frequency. Of particular interest, HIV-1-infected cells with multiple identical integration sites were detected in longitudinal analysis of samples from 3 patients, suggesting that these cells persist for long periods and that clonal expansion may occur. Furthermore, strong integration clusters in the BACH2 gene were observed in 2 patients (31% in patient 1 and 5% in patient 3). Our findings not only raise the possibility of biased target-site integration but also provide mechanistic insights into the long-term persistence of HIV-1.

Impact of V2 mutations on escape from a potent neutralizing anti-V3 monoclonal antibody during in vitro selection of a primary human immunodeficiency virus type 1 isolate.

KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.

Human T cell leukemia virus type I is resistant to the antiviral effects of APOBEC3.

The objective of this study was to investigate whether the anti-retroviral cellular cytidine deaminase, APOBEC3, inhibits the infectivity of human T cell leukemia virus type I (HTLV-I). Sufficient quantities of cell-free HTLV-I virion for infection were obtained by cotransfecting cells with HTLV-I and human or murine APOBEC3 expression vectors along with a plasmid expressing Tax. HTLV-I viruses containing these deaminases were still capable of infecting 293T and MOLT-4 cells. No G-to-A mutations, which are characteristic of cytidine deaminases, were observed in the HTLV-I genome. These results suggest that the enzymatic activity of APOBEC3 may not contribute substantially to antiviral responses to HTLV-I.

Resistance profile of a neutralizing anti-HIV monoclonal antibody, KD-247, that shows favourable synergism with anti-CCR5 inhibitors.

BACKGROUND: The high-affinity humanized monoclonal antibody (MAb) KD-247 reacts with a tip region in gp120-V3 and cross-neutralizes primary isolates with a matching neutralization sequence motif. METHODS: We induced an HIV-1 variant that was resistant to KD-247 by exposing the JR-FL virus to increasing concentrations of KD-247 in PM1/CCR5 cells, which expressed high levels of CCR5 in vitro. We determined the amino acid sequence of the gp120-encoding region of the JR-FL escape mutant from KD-247. To confirm that this substitution was responsible for the KD-247-resistance, a single-round replication assay was performed. We further evaluated the anti-HIV-1 interactions between KD-247 and various CCR5 inhibitors in vitro. RESULTS: At passage 8 of the culture in the presence of 1000 mug/ml KD-247, one amino acid substitution, Gly to Glu at position 314 (G314E), was identified in the V3-tip of gp120. A pseudotyped virus with the G314E mutation was highly resistant to KD-247. Unexpectedly, this mutant virus was sensitive to CCR5 inhibitors, RANTES, recombinant human soluble CD4 (rsCD4) and an anti-CCR5 MAb, but resistant to an anti-CD4 MAb, compared with the wild-type virus. We also found that combinations of KD-247 and CCR5 inhibitors were highly synergistic. CONCLUSIONS: The present data suggest that KD-247 has certain advantages for possible passive immunotherapy. They are: high concentrations of KD-247 are needed for viral acquisition of KD-247 resistance; the escape variants are more sensitive to CCR5 inhibitors and rsCD4; and there are high levels of synergism between KD-247 and CCR5 inhibitors at all concentrations tested.

Fumagillin suppresses HIV-1 infection of macrophages through the inhibition of Vpr activity.

HIV-1 viral protein R (Vpr) is one of the human immunodeficiency virus type 1 encoded proteins that have important roles in viral pathogenesis. However, no clinical drug for AIDS therapy that targets Vpr has been developed. Here, we have established a screening system to isolate Vpr inhibitors using budding yeast cells. We purified a Vpr inhibitory compound from fungal metabolites and identified it as fumagillin, a chemical already known to be a potent inhibitor of angiogenesis. Fumagillin not only reversed the growth inhibitory activity of Vpr in yeast and human cells, but also inhibited Vpr-dependent viral gene expression upon the infection of human macrophages.

Ex vivo neutralization of HIV-1 quasi-species by a broadly reactive humanized monoclonal antibody KD-247.

By immunizing mice sequentially with six different V3 peptides we obtained a murine monoclonal antibody (MAb) C25, and its humanized counterpart KD-247. The MAb recognizes the sequence IGPGRA at the tip of the V3 loop and displays broad neutralizing activity against a variety of HIV-1 isolates. KD-247 was tested in an ex vivo neutralization assay to determine its capability to contain the spread of a quasi species population of clade B HIV-1 derived from two patients. The epitope of KD-247 was generally matching with the V3 sequences of various clones isolated from plasma and peripheral blood mononuclear cells (PBMC) of two patients. Complete or strong inhibition of viral replication was observed when the patients' PBMC were cultured with a high concentration of KD-247. Some neutralization escape variants, which had mutations in the V3 or outside of the V3 loop, emerged only at a low concentration of the MAb. These results suggest that KD-247 could be a good candidate for immunotherapy against HIV-1 in vivo.

Glycoglycerolipid from the membranes of Acholeplasma laidlawii binds to human immunodeficiency virus-1 (HIV-1) and accelerates its entry into cells.

We have reported previously that glycoglycerolipids derived from the membranes of Acholeplasma laidlawii, 3- O-[2'- O-(alpha-D-glucopyranosyl)-6'- O-acyl-alpha-glucopyranosyl]-1,2-di- O-acyl- sn-glycerols (GAGDGs) bind to human cell lines. In addition, the GAGDGs were found to augment HIV-1 infection in human cell lines. Here we show that GAGDG binds to HIV-1 and facilitates the entry of HIV-1 into cells. The binding ability of GAGDG to HIV-1 was blocked by anti-GAGDG serum. Binding assay with synthetic GAGDGs and related compounds showed that the presence of branching form of acyl chains at the C14 or C16 position, glucose, and the acyl chain binding to the glucose were critical for efficient binding. GAGDG efficiently augmented the entry of HIV-1 into cells in a single-cycle replication assay. These results indicate that GAGDG of A. laidlawii membranes participates in the facilitation of HIV-1 infection.

Susceptibility of mink (Mustera vision)-derived cells to replication by human immunodeficiency virus type 1.

In vivo studies for understanding viral transmission and replication, host immune responses, and pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection would greatly benefit from the establishment of a small-animal model. In this study, we explored the potential of American mink (Mustera vison) as a susceptible host. We found that primary cells and cell lines derived from this species efficiently supported trans-activation of the HIV-1 long terminal repeat by Tat. Accordingly, the cysteine residue at position 261, which has been shown to be important for interaction of the human cyclin T1 with the HIV-1 regulatory protein Tat, is conserved in the mink homologue. No species-specific defect in Rev function could be detected in mink cells. In addition, primary splenocytes, fibroblasts, and the Mv.1.Lu cell line from American mink supported early as well as late HIV-1 gene expression following infection with vesicular stomatitis G protein-pseudotyped HIV-1 viruses, at levels comparable to those seen with permissive human cells. Furthermore, the mink Mv.1.Lu cell line stably expressing human CD4 and CCR5 receptors supported a spreading HIV-1 infection with few, if any, deficiencies compared to findings in human cell lines. This indicates the potential of HIV-1 to replicate in these cells once the blockade at the stage of virus entry has been removed. These results clearly show that cells from American mink generally pose no functional intracellular block to HIV-1 replication, and collectively they raise the possibility that this animal species could be engineered to support HIV-1 infection, providing a useful small-animal model for evaluating de novo infection by HIV-1.