Ying Dang

Identification of APOBEC3DE as Another Antiretroviral Factor from the Human APOBEC Family

ABSTRACT A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.

Human cytidine deaminase APOBEC3H restricts HIV-1 replication.

The human genome encodes seven APOBEC3 (A3) cytidine deaminases with potential antiretroviral activity: A3A, A3B, A3C, A3DE, A3F, A3G, and A3H. A3G was the first identified to block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3F, A3B, and A3DE were shown later to have similar activities. HIV-1 produces a protein called Vif that is able to neutralize the antiretroviral activities of A3DE, A3F, and A3G, but not A3B. Only the antiretroviral activity of A3H remains to be defined due to its poor expression in cell culture. Here, we studied the mechanism impairing A3H expression. When primate A3H sequences were compared, a premature termination codon was identified on the fifth exon of the human and chimpanzee A3H genes, which significantly decreased their protein expression. It causes a 29-residue deletion from the C terminus, and this truncation did not reduce human A3H protein stability. However, the mRNA levels of the truncated gene were significantly decreased. Human A3H protein expression could be restored to a normal level either by repairing this truncation or through expression from a vector containing an intron from human cytomegalovirus. Once expression was optimized, human A3H could reduce HIV-1 infectivity up to 150-fold. Importantly, HIV-1 Vif failed to neutralize A3H activity. Nevertheless, extensive sequence analysis could not detect any significant levels of G-to-A mutation in the HIV-1 genome by human A3H. Thus, A3H inhibits HIV-1 replication potently by a cytidine deamination-independent mechanism, and optimizing A3H expression in vivo should represent a novel therapeutic strategy for HIV-1 treatment.

Identification of a Novel WxSLVK Motif in the N Terminus of Human Immunodeficiency Virus and Simian Immunodeficiency Virus Vif That Is Critical for APOBEC3G and APOBEC3F Neutralization

ABSTRACT The function of lentiviral Vif proteins is to neutralize the host antiviral cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F). Vif bridges a cullin 5-based E3 ubiquitin ligase with A3G and A3F and mediates their degradation by proteasomes. Recent studies have found that Vif uses different domains to bind to A3G and A3F. A 14DRMR17 domain binds to A3F, 40YRHHY44 binds to A3G, and 69YxxL72 binds to both A3G and A3F. Here, we report another functional domain of Vif. Previously, we demonstrated that human immunodeficiency virus type 1 (HIV-1) Vif failed to mediate A3G proteasomal degradation when all 16 lysines were mutated to arginines. Here, we show that K26, and to a lesser extent K22, is critical for A3G neutralization. K22 and K26 are part of a conserved 21WxSLVK26 (x represents N, K, or H) motif that is found in most primate lentiviruses and that shows species-specific variation. Both K22 and K26 in this motif regulated Vif specificity only for A3G, whereas the SLV residues regulated Vif specificity for both A3F and A3G. Interestingly, SLV and K26 in HIV-1 Vif did not directly mediate Vif interaction with either A3G or A3F. Previously, other groups have reported an important role for W21 in A3F and A3G neutralization. Thus, 21WxSLVK26 is a novel functional domain that regulates Vif activity toward both A3F and A3G and is a potential drug target to inhibit Vif activity and block HIV-1 replication.

Moloney Leukemia Virus 10 (MOV10) Protein Inhibits Retrovirus Replication

Moloney leukemia virus 10 (MOV10) protein is a superfamily-1 RNA helicase, and it is also a component of the RNA-induced silencing complex. Recent studies have shown that MOV10 plays an active role in the RNA interference pathway. Here, we report that MOV10 inhibits retrovirus replication. When it was overexpressed in viral producer cells, MOV10 was able to reduce the infectivity of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and murine leukemia virus. Conversely, when MOV10 expression was reduced by small interfering RNAs, HIV-1 infectivity was increased. Consistently, silencing of MOV10 expression in a human T cell line enhanced HIV-1 replication. Furthermore, we found that MOV10 interacts with HIV-1 nucleocapsid protein in an RNA-dependent manner and is packaged into virions. It blocks HIV-1 replication at a postentry step. In addition, we also found that HIV-1 could suppress MOV10 protein expression to counteract this cellular resistance. All of these results indicate that MOV10 has a broad antiretroviral activity that can target a wide range of retroviruses, and it could be actively involved in host defense against retroviral infection.

Analysis of Human APOBEC3H Haplotypes and Anti-Human Immunodeficiency Virus Type 1 Activity

ABSTRACT Human APOBEC3H (A3H) has one cytidine deaminase domain (CDD) and inhibits the replication of retrotransposons and human immunodeficiency virus type 1 (HIV-1) in a Vif-resistant manner. Human A3H has five single amino acid polymorphisms (N15Δ, R18L, G105R, K121D, and E178D), and four haplotypes (I to IV) have previously been identified in various human populations. Haplotype II was primarily found in African-derived populations, and it was the only one that could be stably expressed. Here, we identified three new haplotypes from six human population samples, which we have named V, VI, and VII. Haplotypes V and VII are stably expressed and inhibit HIV-1 replication. Notably, haplotype V was identified in samples from all African-, Asian-, and Caucasian-derived populations studied. Using haplotype VII, we investigated the A3H anti-HIV-1 mechanism. We found that A3H virion packaging is independent of its CDD but dependent on a 112YYXW115 motif. This motif binds HIV-1 nucleocapsid in an RNA-dependent manner, and a single Y112A mutation completely disrupts A3H virion incorporation. We further studied the mechanism of A3H resistance to Vif. Although the previously identified APOBEC3G Vif-responsive motif 128DPDY131 is not conserved in A3H, placement of this motif into A3H does not make it become less resistant to HIV-1 Vif. We conclude that stably expressed A3H haplotypes may be more broadly distributed in humans than previously realized, and A3H protein is resistant to Vif. These results have important implications for the role of A3H in retrotransposon and HIV-1 inhibition.

Biochemical differentiation of APOBEC3F and APOBEC3G proteins associated with HIV-1 life cycle.

APOBEC3G and APOBEC3F are cytidine deaminase with duplicative cytidine deaminase motifs that restrict HIV-1 replication by catalyzing C-to-U transitions on nascent viral cDNA. Despite 60% protein sequence similarity, APOBEC3F and APOBEC3G have a different target consensus sequence for editing, and importantly, APOBEC3G has 10-fold higher anti-HIV activity than APOBEC3F. Thus, APOBEC3F and APOBEC3G may have distinctive characteristics that account for their functional differences. Here, we have biochemically characterized human APOBEC3F and APOBEC3G protein complexes as a function of the HIV-1 life cycle. APOBEC3G was previously shown to form RNase-sensitive, enzymatically inactive, high molecular mass complexes in immortalized cells, which are converted into enzymatically active, low molecular mass complexes by RNase digestion. We found that APOBEC3F also formed high molecular mass complexes in these cells, but these complexes were resistant to RNase treatment. Further, the N-terminal half determined RNase sensitivity and was necessary for the high molecular mass complex assembly of APOBEC3G but not APOBEC3F. Unlike APOBEC3F, APOBEC3G strongly interacted with cellular proteins via disulfide bonds. Inside virions, both APOBEC3F and APOBEC3G were found in viral cores, but APOBEC3G was associated with low molecular mass, whereas APOBEC3F was still retained in high molecular mass complexes. After cell entry, both APOBEC3F and APOBEC3G were localized in low molecular mass complexes associated with viral reverse transcriptional machinery. These results demonstrate that APOBEC3F and APOBEC3G complexes undergo dynamic conversion during HIV-1 infection and also reveal biochemical differences that likely determine their different anti-HIV-1 activity.

APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated

Although the covalent attachment of a polyubiquitin is the prevailing paradigm for entry into proteasomes, accumulating evidence suggests that poorly defined ubiquitin-free pathways also degrade proteins. The cytidine deaminase APOBEC3G (A3G) potently inhibits human immunodeficiency virus type 1 replication by disrupting viral reverse transcription. However, human immunodeficiency virus type 1 produces a viral infectivity factor (Vif) to destroy this antiretroviral protein. It was shown that Vif binds to both A3G and a Cullin 5 ubiquitin-protein isopeptide ligase. It is currently accepted that this enzyme polyubiquitylates A3G on lysine residues, resulting in its degradation by proteasomes. Here, we find that A3G without ubiquitylation is still degraded by proteasomes in a Vif-dependent manner. We further show that Vif is polyubiquitylated and that this event could be critical for A3G proteasomal degradation. Thus, A3G is degraded by a novel pathway that might involve ubiquitylation of one protein and then targets a second binding partner for proteasomal entry and degradation. We propose that instead of triggering A3G polyubiquitylation, polyubiquitylated Vif might serve as a vehicle to transport A3G into proteasomes for degradation.

Identification of 81LGxGxxIxW89 and 171EDRW174 Domains from Human Immunodeficiency Virus Type 1 Vif That Regulate APOBEC3G and APOBEC3F Neutralizing Activity

ABSTRACT The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, 81LGxGxSIEW89 and 171EDRWN175. Within the 81LGxGxSIEW89 sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues 86SIEW89 affect Vif binding to A3F, A3G, and Cul5. Accordingly, this 81LGxGxSIEW89 sequence was designated the 81LGxGxxIxW89 domain. Within the 171EDRWN175 sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated 171EDRW174. The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, 81LGxGxxIxW89 and 171EDRW174 are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.

Identification of a critical T(Q/D/E)x5ADx2(I/L) motif from primate lentivirus Vif proteins that regulate APOBEC3G and APOBEC3F neutralizing activity.

ABSTRACT Primate lentiviruses are unique in that they produce several accessory proteins to help in the establishment of productive viral infection. The major function of these proteins is to clear host resistance factors that inhibit viral replication. Vif is one of these proteins. It functions as an adaptor that binds to the cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) and bridges them to a cullin 5 (Cul5) and elongin (Elo) B/C E3 ubiquitin ligase complex for proteasomal degradation. So far, 11 discontinuous domains in Vif have been identified that regulate this degradation process. Here we report another domain, T(Q/D/E)x5ADx2(I/L), which is located at residues 96 to 107 in the human immunodeficiency virus type 1 (HIV-1) Vif protein. This domain is conserved not only in all HIV-1 subtypes but also in other primate lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV), which infects rhesus macaques (SIVmac) and African green monkeys (SIVagm). Mutations of the critical residues in this motif seriously disrupted Vifs neutralizing activity toward both A3G and A3F. This motif regulates Vif interaction not only with A3G and A3F but also with Cul5. When this motif was inactivated in the HIV-1 genome, Vif failed to exclude A3G and A3F from virions, resulting in abortive HIV replication in nonpermissive human T cells. Thus, T(Q/D/E)x5ADx2(I/L) is a critical functional motif that directly supports the adaptor function of Vif and is an attractive target for inhibition of Vif function.

APOBEC3G and APOBEC3F Require an Endogenous Cofactor to Block HIV-1 Replication

APOBEC3G (A3G)/APOBEC3F (A3F) are two members of APOBEC3 cytidine deaminase subfamily. Although they potently inhibit the replication of vif-deficient HIV-1, this mechanism is still poorly understood. Initially, A3G/A3F were thought to catalyze C-to-U transitions on the minus-strand viral cDNAs during reverse transcription to disrupt the viral life cycle. Recently, it was found more likely that A3G/A3F directly interrupts viral reverse transcription or integration. In addition, A3G/A3F are both found in the high-molecular-mass complex in immortalized cell lines, where they interact with a number of different cellular proteins. However, there has been no evidence to prove that these interactions are required for A3G/A3F function. Here, we studied A3G/A3F-restricted HIV-1 replication in six different human T cell lines by infecting them with wild-type or vif-deficient HIV-1. Interestingly, in a CEM-derived cell line CEM-T4, which expresses high levels of A3G/A3F proteins, the vif-deficient virus replicated as equally well as the wild-type virus, suggesting that these endogenous antiretroviral genes lost anti-HIV activities. It was confirmed that these A3G/A3F genes do not contain any mutation and are functionally normal. Consistently, overexpression of exogenous A3G/A3F in CEM-T4 cells still failed to restore their anti-HIV activities. However, this activity could be restored if CEM-T4 cells were fused to 293T cells to form heterokaryons. These results demonstrate that CEM-T4 cells lack a cellular cofactor, which is critical for A3G/A3F anti-HIV activity. We propose that a further study of this novel factor will provide another strategy for a complete understanding of the A3G/A3F antiretroviral mechanism.