The cyclophilin A-binding loop of the capsid regulates the human TRIM5α sensitivity of nonpandemic HIV-1.

The rather few cases of humans infected by HIV-1 N, O, or P raise the question of their incomplete adaptation to humans. We hypothesized that early postentry restrictions may be relevant for the impaired spread of these HIVs. One of the best-characterized species-specific restriction factors is TRIM5α. HIV-1 M can escape human (hu) TRIM5α restriction by binding cyclophilin A (CYPA, also known as PPIA, peptidylprolyl isomerase A) to the so-called CYPA-binding loop of its capsid protein. How non-M HIV-1s interact with huTRIM5α is ill-defined. By testing full-length reporter viruses (Δ env) of HIV-1 N, O, P, and SIVgor (simian IV of gorillas), we found that in contrast to HIV-1 M, the nonpandemic HIVs and SIVgor showed restriction by huTRIM5α. Work to identify capsid residues that mediate susceptibility to huTRIM5α revealed that residue 88 in the capsid CYPA-binding loop was important for such differences. There, HIV-1 M uses alanine to resist, while non-M HIV-1s have either valine or methionine, which avail them for huTRIM5α. Capsid residue 88 determines the sensitivity to TRIM5α in an unknown way. Molecular simulations indicated that capsid residue 88 can affect trans-to-cis isomerization patterns on the capsids of the viruses we tested. These differential CYPA usages by pandemic and nonpandemic HIV-1 suggest that the enzymatic activity of CYPA on the viral core might be important for its protective function against huTRIM5α.

HIV-1 restriction by SERINC5.

Serine incorporator 5 (SERINC5 or SER5) is a multipass transmembrane protein with ill-defined cellular activities. SER5 was recently described as a human immunodeficiency virus 1 (HIV-1) restriction factor capable of inhibiting HIV-1 that does not express its accessory protein Nef (Δ Nef). SER5 incorporated into the viral membrane impairs the entry of HIV-1 by disrupting the fusion between the viral and the plasma membrane after envelope receptor interaction induced the first steps of the fusion process. The mechanisms of how SER5 prevents membrane fusion are not fully understood and viral envelope proteins were identified that escape the SER5-mediated restriction. Primate lentiviruses, such as HIV-1 and simian immunodeficiency viruses (SIVs), use their accessory protein Nef to downregulate SER5 from the plasma membrane by inducing an endocytic pathway. In addition to being directly antiviral, recent data suggest that SER5 is an important adapter protein in innate signaling pathways leading to the induction of inflammatory cytokines. This review discusses the current knowledge about HIV-1 restriction by SER5.

Cell Type-Dependent Escape of Capsid Inhibitors by Simian Immunodeficiency Virus SIVcpz.

Pandemic human immunodeficiency virus type 1 (HIV-1) is the result of the zoonotic transmission of simian immunodeficiency virus (SIV) from the chimpanzee subspecies Pan troglodytestroglodytes (SIVcpzPtt). The related subspecies Pan troglodytesschweinfurthii is the host of a similar virus, SIVcpzPts, which did not spread to humans. We tested these viruses with small-molecule capsid inhibitors (PF57, PF74, and GS-CA1) that interact with a binding groove in the capsid that is also used by CPSF6. While HIV-1 was sensitive to capsid inhibitors in cell lines, human macrophages, and peripheral blood mononuclear cells (PBMCs), SIVcpzPtt was resistant in rhesus FRhL-2 cells and human PBMCs but was sensitive to PF74 in human HOS and HeLa cells. SIVcpzPts was insensitive to PF74 in FRhL-2 cells, HeLa cells, PBMCs, and macrophages but was inhibited by PF74 in HOS cells. A truncated version of CPSF6 (CPSF6-358) inhibited SIVcpzPtt and HIV-1, while in contrast, SIVcpzPts was resistant to CPSF6-358. Homology modeling of HIV-1, SIVcpzPtt, and SIVcpzPts capsids and binding energy estimates suggest that these three viruses bind similarly to the host proteins cyclophilin A (CYPA) and CPSF6 as well as the capsid inhibitor PF74. Cyclosporine treatment, mutation of the CYPA-binding loop in the capsid, or CYPA knockout eliminated the resistance of SIVcpzPts to PF74 in HeLa cells. These experiments revealed that the antiviral capacity of PF74 is controlled by CYPA in a virus- and cell type-specific manner. Our data indicate that SIVcpz viruses can use infection pathways that escape the antiviral activity of PF74. We further suggest that the antiviral activity of PF74 capsid inhibitors depends on cellular cofactors.IMPORTANCE HIV-1 originated from SIVcpzPtt but not from the related virus SIVcpzPts, and thus, it is important to describe molecular infection by SIVcpzPts in human cells to understand the zoonosis of SIVs. Pharmacological HIV-1 capsid inhibitors (e.g., PF74) bind a capsid groove that is also a binding site for the cellular protein CPSF6. SIVcpzPts was resistant to PF74 in HeLa cells but sensitive in HOS cells, thus indicating cell line-specific resistance. Both SIVcpz viruses showed resistance to PF74 in human PBMCs. Modulating the presence of cyclophilin A or its binding to capsid in HeLa cells overcame SIVcpzPts resistance to PF74. These results indicate that early cytoplasmic infection events of SIVcpzPts may differ between cell types and affect, in an unknown manner, the antiviral activity of capsid inhibitors. Thus, capsid inhibitors depend on the activity or interaction of currently uncharacterized cellular factors.

Identification of a Conserved Interface of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Vifs with Cullin 5.

Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif.IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.

USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1.

The host intrinsic innate immune system drives antiviral defenses and viral restriction, which includes the production of soluble factors, such as type I and III interferon (IFN), and activation of restriction factors, including SAMHD1, a deoxynucleoside triphosphohydrolase. Interferon-stimulated gene 15 (ISG15)-specific ubiquitin-like protease 43 (USP18) abrogates IFN signaling pathways. The cyclin-dependent kinase inhibitor p21 (CIP1/WAF1), which is involved in the differentiation and maturation of monocytes, inhibits human immunodeficiency virus type 1 (HIV-1) in macrophages and dendritic cells. p21 inhibition of HIV-1 replication is thought to occur at the reverse transcription step, likely by suppressing cellular deoxynucleoside triphosphate (dNTP) biosynthesis and increasing the amount of antivirally active form of SAMHD1. SAMHD1 strongly inhibits HIV-1 replication in myeloid and resting CD4+ T cells. Here, we studied how USP18 influences HIV-1 replication in human myeloid THP-1 cells. We found that USP18 has the novel ability to inhibit the antiviral function of p21 in differentiated THP-1 cells. USP18 enhanced reverse transcription of HIV-1 by downregulating p21 expression and upregulating intracellular dNTP levels. p21 downregulation by USP18 was associated with the active form of SAMHD1, phosphorylated at T592. USP18 formed a complex with the E3 ubiquitin ligase recognition factor SKP2 (S-phase kinase associated protein 2) and SAMHD1. CRISPR-Cas9 knockout of USP18 increased p21 protein expression and blocked HIV-1 replication. Overall, we propose USP18 as a regulator of p21 antiviral function in differentiated myeloid THP-1 cells.IMPORTANCE Macrophages and dendritic cells are usually the first point of contact with pathogens, including lentiviruses. Host restriction factors, including SAMHD1, mediate the innate immune response against these viruses. However, HIV-1 has evolved to circumvent the innate immune response and establishes disseminated infection. The cyclin-dependent kinase inhibitor p21, which is involved in differentiation and maturation of monocytes, blocks HIV-1 replication at the reverse transcription step. p21 is thought to suppress key enzymes involved in dNTP biosynthesis and activates SAMHD1 antiviral function. We report here that the human USP18 protein is a novel factor potentially contributing to HIV replication by blocking the antiviral function of p21 in differentiated human myeloid cells. USP18 downregulates p21 protein expression, which correlates with upregulated intracellular dNTP levels and the antiviral inactive form of SAMHD1. Depletion of USP18 stabilizes p21 protein expression, which correlates with dephosphorylated SAMHD1 and a block to HIV-1 replication.

Stably expressed APOBEC3H forms a barrier for cross-species transmission of simian immunodeficiency virus of chimpanzee to humans.

APOBEC3s (A3s) are potent restriction factors of human immunodeficiency virus type 1/simian immunodeficiency viruses (HIV-1/SIV), and can repress cross-species transmissions of lentiviruses. HIV-1 originated from a zoonotic infection of SIV of chimpanzee (SIVcpz) to humans. However, the impact of human A3s on the replication of SIVcpz remains unclear. By using novel SIVcpz reporter viruses, we identified that human APOBEC3B (A3B) and APOBEC3H (A3H) haplotype II strongly reduced the infectivity of SIVcpz, because both of them are resistant to SIVcpz Vifs. We further demonstrated that human A3H inhibited SIVcpz by deaminase dependent as well independent mechanisms. In addition, other stably expressed human A3H haplotypes and splice variants showed strong antiviral activity against SIVcpz. Moreover, most SIV and HIV lineage Vif proteins could degrade chimpanzee A3H, but no Vifs from SIVcpz and SIV of gorilla (SIVgor) lineages antagonized human A3H haplotype II. Expression of human A3H hapII in human T cells efficiently blocked the spreading replication of SIVcpz. The spreading replication of SIVcpz was also restricted by stable A3H in human PBMCs. Thus, we speculate that stably expressed human A3H protects humans against the cross-species transmission of SIVcpz and that SIVcpz spillover to humans may have started in individuals that harbor haplotypes of unstable A3H proteins.

Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential.

BACKGROUND: Hosts are able to restrict viral replication to contain virus spread before adaptive immunity is fully initiated. Many viruses have acquired genes directly counteracting intrinsic restriction mechanisms. This phenomenon has led to a co-evolutionary signature for both the virus and host which often provides a barrier against interspecies transmission events. Through different mechanisms of action, but with similar consequences, spumaviral feline foamy virus (FFV) Bet and lentiviral feline immunodeficiency virus (FIV) Vif counteract feline APOBEC3 (feA3) restriction factors that lead to hypermutation and degradation of retroviral DNA genomes. Here we examine the capacity of vif to substitute for bet function in a chimeric FFV to assess the transferability of anti-feA3 factors to allow viral replication. RESULTS: We show that vif can replace bet to yield replication-competent chimeric foamy viruses. An in vitro selection screen revealed that an engineered Bet-Vif fusion protein yields suboptimal protection against feA3. After multiple passages through feA3-expressing cells, however, variants with optimized replication competence emerged. In these variants, Vif was expressed independently from an N-terminal Bet moiety and was stably maintained. Experimental infection of immunocompetent domestic cats with one of the functional chimeras resulted in seroconversion against the FFV backbone and the heterologous FIV Vif protein, but virus could not be detected unambiguously by PCR. Inoculation with chimeric virus followed by wild-type FFV revealed that repeated administration of FVs allowed superinfections with enhanced antiviral antibody production and detection of low level viral genomes, indicating that chimeric virus did not induce protective immunity against wild-type FFV. CONCLUSIONS: Unrelated viral antagonists of feA3 cellular restriction factors can be exchanged in FFV, resulting in replication competence in vitro that was attenuated in vivo. Bet therefore may have additional functions other than A3 antagonism that are essential for successful in vivo replication. Immune reactivity was mounted against the heterologous Vif protein. We conclude that Vif-expressing FV vaccine vectors may be an attractive tool to prevent or modulate lentivirus infections with the potential option to induce immunity against additional lentivirus antigens.

Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s.

Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain. IMPORTANCE: Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here, we identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z2 or APOBEC3Z3. These specific Vif sites are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in FIV Vif from puma. Our findings provide important insights for future experiments describing the FIV Vif interaction with feline APOBEC3s and also indicate that the conserved feline APOBEC3s interaction sites of FIV Vif allow FIV transmissions in Felidae.

A Naturally Occurring Domestic Cat APOBEC3 Variant Confers Resistance to Feline Immunodeficiency Virus Infection.

UNLABELLED: Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) DNA cytosine deaminases can be incorporated into progeny virions and inhibit lentiviral replication. On the other hand, viral infectivity factor (Vif) of lentiviruses antagonizes A3-mediated antiviral activities by degrading A3 proteins. It is known that domestic cat (Felis catus) APOBEC3Z3 (A3Z3), the ortholog of human APOBEC3H, potently suppresses the infectivity of vif-defective feline immunodeficiency virus (FIV). Although a recent report has shown that domestic cat encodes 7 haplotypes (hap I to hap VII) of A3Z3, the relevance of A3Z3 polymorphism in domestic cats with FIV Vif has not yet been addressed. In this study, we demonstrated that these feline A3Z3 variants suppress vif-defective FIV infectivity. We also revealed that codon 65 of feline A3Z3 is a positively selected site and that A3Z3 hap V is subject to positive selection during evolution. It is particularly noteworthy that feline A3Z3 hap V is resistant to FIV Vif-mediated degradation and still inhibits vif-proficient viral infection. Moreover, the side chain size, but not the hydrophobicity, of the amino acid at position 65 determines the resistance to FIV Vif-mediated degradation. Furthermore, phylogenetic analyses have led to the inference that feline A3Z3 hap V emerged approximately 60,000 years ago. Taken together, these findings suggest that feline A3Z3 hap V may have been selected for escape from an ancestral FIV. This is the first evidence for an evolutionary "arms race" between the domestic cat and its cognate lentivirus. IMPORTANCE: Gene diversity and selective pressure are intriguing topics in the field of evolutionary biology. A direct interaction between a cellular protein and a viral protein can precipitate an evolutionary arms race between host and virus. One example is primate APOBEC3G, which potently restricts the replication of primate lentiviruses (e.g., human immunodeficiency virus type 1 [HIV-1] and simian immunodeficiency virus [SIV]) if its activity is not counteracted by the viral Vif protein. Here we investigate the ability of 7 naturally occurring variants of feline APOBEC3, APOBEC3Z3 (A3Z3), to inhibit FIV replication. Interestingly, one feline A3Z3 variant is dominant, restrictive, and naturally resistant to FIV Vif-mediated degradation. Phylogenetic analyses revealed that the ancestral change that generated this variant could have been caused by positive Darwinian selection, presumably due to an ancestral FIV infection. The experimental-phylogenetic investigation sheds light on the evolutionary history of the domestic cat, which was likely influenced by lentiviral infection.

Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C.

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F. IMPORTANCE: The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors.

BACKGROUND: Feline immunodeficiency virus (FIV) is a global pathogen of Felidae species and a model system for Human immunodeficiency virus (HIV)-induced AIDS. In felids such as the domestic cat (Felis catus), APOBEC3 (A3) genes encode for single-domain A3Z2s, A3Z3 and double-domain A3Z2Z3 anti-viral cytidine deaminases. The feline A3Z2Z3 is expressed following read-through transcription and alternative splicing, introducing a previously untranslated exon in frame, encoding a domain insertion called linker. Only A3Z3 and A3Z2Z3 inhibit Vif-deficient FIV. Feline A3s also are restriction factors for HIV and Simian immunodeficiency viruses (SIV). Surprisingly, HIV-2/SIV Vifs can counteract feline A3Z2Z3. RESULTS: To identify residues in feline A3s that Vifs need for interaction and degradation, chimeric human-feline A3s were tested. Here we describe the molecular direct interaction of feline A3s with Vif proteins from cat FIV and present the first structural A3 model locating these interaction regions. In the Z3 domain we have identified residues involved in binding of FIV Vif, and their mutation blocked Vif-induced A3Z3 degradation. We further identified additional essential residues for FIV Vif interaction in the A3Z2 domain, allowing the generation of FIV Vif resistant A3Z2Z3. Mutated feline A3s also showed resistance to the Vif of a lion-specific FIV, indicating an evolutionary conserved Vif-A3 binding. Comparative modelling of feline A3Z2Z3 suggests that the residues interacting with FIV Vif have, unlike Vif-interacting residues in human A3s, a unique location at the domain interface of Z2 and Z3 and that the linker forms a homeobox-like domain protruding of the Z2Z3 core. HIV-2/SIV Vifs efficiently degrade feline A3Z2Z3 by possible targeting the linker stretch connecting both Z-domains. CONCLUSIONS: Here we identified in feline A3s residues important for binding of FIV Vif and a unique protein domain insertion (linker). To understand Vif evolution, a structural model of the feline A3 was developed. Our results show that HIV Vif binds human A3s differently than FIV Vif feline A3s. The linker insertion is suggested to form a homeo-box domain, which is unique to A3s of cats and related species, and not found in human and mouse A3s. Together, these findings indicate a specific and different A3 evolution in cats and human.

Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity.

LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.

Vif determines the requirement for CBF-ß in APOBEC3 degradation.

APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular DNA deaminases that restrict a broad spectrum of lentiviruses. This process is counteracted by Vif (viral infectivity factor) of lentiviruses, which binds APOBEC3s and promotes their degradation. CBF-ß (core binding factor subunit ß) is an essential co-factor for the function of human immunodeficiency virus type 1 Vif to degrade human APOBEC3s. However, the requirement for CBF-ß in Vif-mediated degradation of other mammalian APOBEC3 proteins is less clear. Here, we determined the sequence of feline CBFB and performed phylogenetic analyses. These analyses revealed that mammalian CBFB is under purifying selection. Moreover, we demonstrated that CBF-ß is dispensable for feline immunodeficiency virus Vif-mediated degradation of APOBEC3s of its host. These findings suggested that primate lentiviruses have adapted to use CBF-ß, an evolutionary stable protein, to counteract APOBEC3 proteins of their hosts after diverging from other lentiviruses.

A functional conserved intronic G run in HIV-1 intron 3 is critical to counteract APOBEC3G-mediated host restriction.

BACKGROUND: The HIV-1 accessory proteins, Viral Infectivity Factor (Vif) and the pleiotropic Viral Protein R (Vpr) are important for efficient virus replication. While in non-permissive cells an appropriate amount of Vif is critical to counteract APOBEC3G-mediated host restriction, the Vpr-induced G2 arrest sets the stage for highest transcriptional activity of the HIV-1 long terminal repeat. RESULTS: We identified a G run localized deep in the vpr AUG containing intron 3 (GI3-2), which was critical for balanced splicing of both vif and vpr non-coding leader exons. Inactivation of GI3-2 resulted in excessive exon 3 splicing as well as exon-definition mediated vpr mRNA formation. However, in an apparently mutually exclusive manner this was incompatible with recognition of upstream exon 2 and vif mRNA processing. As a consequence, inactivation of GI3-2 led to accumulation of Vpr protein with a concomitant reduction in Vif protein. We further demonstrate that preventing hnRNP binding to intron 3 by GI3-2 mutation diminished levels of vif mRNA. In APOBEC3G-expressing but not in APOBEC3G-deficient T cell lines, mutation of GI3-2 led to a considerable replication defect. Moreover, in HIV-1 isolates carrying an inactivating mutation in GI3-2, we identified an adjacent G-rich sequence (GI3-1), which was able to substitute for the inactivated GI3-2. CONCLUSIONS: The functionally conserved intronic G run in HIV-1 intron 3 plays a major role in the apparently mutually exclusive exon selection of vif and vpr leader exons and hence in vif and vpr mRNA formation. The competition between these exons determines the ability to evade APOBEC3G-mediated antiviral effects due to optimal vif expression.

Prototype foamy virus Bet impairs the dimerization and cytosolic solubility of human APOBEC3G.

Cellular cytidine deaminases from the APOBEC3 family are potent restriction factors that are able to block the replication of retroviruses. Consequently, retroviruses have evolved a variety of different mechanisms to counteract inhibition by APOBEC3 proteins. Lentiviruses such as human immunodeficiency virus (HIV) express Vif, which interferes with APOBEC3 proteins by targeting these restriction factors for proteasomal degradation, hence blocking their ability to access the reverse transcriptase complex in the virions. Other retroviruses use less-well-characterized mechanisms to escape the APOBEC3-mediated cellular defense. Here we show that the prototype foamy virus Bet protein can protect foamy viruses and an unrelated simian immunodeficiency virus against human APOBEC3G (A3G). In our system, Bet binds to A3G and prevents its encapsidation without inducing its degradation. Bet failed to coimmunoprecipitate with A3G mutants unable to form homodimers and dramatically reduced the recovery of A3G proteins from soluble cytoplasmic cell fractions. The Bet-A3G interaction is probably a direct binding interaction and seems to be independent of RNA. Together, these data suggest a novel model whereby Bet uses two possibly complementary mechanisms to counteract A3G: (i) Bet prevents encapsidation of A3G by blocking A3G dimerization, and (ii) Bet sequesters A3G in immobile complexes, impairing its ability to interact with nascent virions.

An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA.

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

The ISG15-Protease USP18 Is a Pleiotropic Enhancer of HIV-1 Replication.

The innate immune response to viruses is formed in part by interferon (IFN)-induced restriction factors, including ISG15, p21, and SAMHD1. IFN production can be blocked by the ISG15-specific protease USP18. HIV-1 has evolved to circumvent host immune surveillance. This mechanism might involve USP18. In our recent studies, we demonstrate that HIV-1 infection induces USP18, which dramatically enhances HIV-1 replication by abrogating the antiviral function of p21. USP18 downregulates p21 by accumulating misfolded dominant negative p53, which inactivates wild-type p53 transactivation, leading to the upregulation of key enzymes involved in de novo dNTP biosynthesis pathways and inactivated SAMHD1. Despite the USP18-mediated increase in HIV-1 DNA in infected cells, it is intriguing to note that the cGAS-STING-mediated sensing of the viral DNA is abrogated. Indeed, the expression of USP18 or knockout of ISG15 inhibits the sensing of HIV-1. We demonstrate that STING is ISGylated at residues K224, K236, K289, K347, K338, and K370. The inhibition of STING K289-linked ISGylation suppresses its oligomerization and IFN induction. We propose that human USP18 is a novel factor that potentially contributes in multiple ways to HIV-1 replication.

Identification of the feline foamy virus Bet domain essential for APOBEC3 counteraction.

BACKGROUND: APOBEC3 (A3) proteins restrict viral replication by cytidine deamination of viral DNA genomes and impairing reverse transcription and integration. To escape this restriction, lentiviruses have evolved the viral infectivity factor (Vif), which binds A3 proteins and targets them for proteolytic degradation. In contrast, foamy viruses (FVs) encode Bet proteins that allow replication in the presence of A3, apparently by A3 binding and/or sequestration, thus preventing A3 packaging into virions and subsequent restriction. Due to a long-lasting FV-host coevolution, Bet proteins mainly counteract restriction by A3s from their cognate or highly related host species. RESULTS: Through bioinformatics, we identified conserved motifs in Bet, all localized in the bel2 exon. In line with the localization of these conserved motifs within bel2, this part of feline FV (FFV) Bet has been shown to be essential for feline A3 (feA3) inactivation and feA3 protein binding. To study the function of the Bet motifs in detail, we analyzed the ability of targeted deletion, substitution, and chimeric FFV-PFV (prototype FV) Bet mutants to physically bind and/or inactivate feA3. Binding of Bet to feA3Z2b is sensitive to mutations in the first three conserved motifs and N- and C-terminal deletions and substitutions across almost the complete bel2 coding sequence. In contrast, the Bel1 (also designated Tas) domain of Bet is dispensable for basal feA3Z2b inactivation and binding but mainly increases the steady state level of Bet. Studies with PFV Bel1 and full-length FFV Bel2 chimeras confirmed the importance of Bel2 for A3 inactivation indicating that Bel1 is dispensable for basal feA3Z2b inactivation and binding but increases Bet stability. Moreover, the bel1/tas exon may be required for expression of a fully functional Bet protein from a spliced transcript. CONCLUSIONS: We show that the Bel2 domain of FV Bet is essential for the inactivation of APOBEC3 cytidine deaminase restriction factors. The Bel1/Tas domain increases protein stability and can be exchanged by related sequence. Since feA3 binding and inactivation by Bet are highly correlated, the data support the view that FV Bet prevents A3-mediated restriction of viral replication by creating strong complexes with these proteins.

Structural features of antiviral DNA cytidine deaminases.

The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans. All A3 proteins can deaminate cytidines to uridines in single-stranded DNA (ssDNA), generated during viral reverse transcription. A3 proteins have either one or two cytidine deaminase domains (CD). The CDs coordinate a zinc ion, and their amino acid specificity classifies the A3s into A3Z1, A3Z2, and A3Z3. A3 proteins occur as monomers, dimers, and large oligomeric complexes. Studies on the nature of A3 oligomerization, as well as the mode of interaction of A3s with RNA and ssDNA are partially controversial. High-resolution structures of the catalytic CD2 of A3G and A3F as well as of the single CD proteins A3A and A3C have been published recently. The NMR and X-ray crystal structures show globular proteins with six α-helices and five ß sheets arranged in a characteristic motif (α1-ß1-ß2/2'-α2-ß3-α3-ß4-α4-ß5-α5-α6). However, the detailed arrangement and extension of individual structure elements and their relevance for A3 complex formation and activity remains a matter of debate and will be highlighted in this review.

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1.

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.