Vpr14-88-Apobec3G Fusion Protein Is Efficiently Incorporated into Vif-Positive HIV-1 Particles and Inhibits Viral Infection

Ao, Zhujun; Yu, Zhe; Wang, Lina; Zheng, Yingfeng; Yao, Xiaojian

doi:10.1371/journal.pone.0001995

Cited by 32 publications

(44 citation statements)

References 68 publications

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“…Since the major function of HIV-1 Vif is to exclude A3G from HIV-1 virions, enhancement of A3G viral incorporation will have a significant impact on anti-AIDS pharmacologic strategies. Indeed, when A3G manages to be packaged into HIV-1 virions through upregulation of expression, the exosomal pathway, enhancement of packaging, or resistance to Vif-induced degradation, it can inhibit the replication of even Vif-proficient HIV-1 (1,4,13,31,33,57,68). Here, we showed that lysinedeficient A3G (A3G20K/R) was resistant to Vif-induced degradation when an HA tag was fused to the N terminus of A3G20K/R (Fig.…”

Section: Discussionmentioning

confidence: 79%

“…At 0, 8, 24, and 48 h postinfection, equal aliquots of cells were collected and washed with phosphate-buffered saline (PBS), and total cellular DNA was isolated from the cells with a DNeasy DNA isolation kit (Qiagen). The primers for the detection of early reverse transcription (ERT), late reverse transcription (LRT), and glyceraldehyde-3-phosphate dehydrogenase have been described elsewhere (4,32,55). Real-time PCR assays were performed on an iCycler instrument with iQ Sybr green Supermix (Bio-Rad).…”

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confidence: 99%

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N-Terminal Hemagglutinin Tag Renders Lysine-Deficient APOBEC3G Resistant to HIV-1 Vif-Induced Degradation by Reduced Polyubiquitination

Wang

Shao

et al. 2011

J Virol

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APOBEC3G, a potent HIV-1 host restriction factor, is overcome by HIV-1 viral infectivity factor (Vif), which induces its polyubiquitination and proteasomal degradation. Here we show that lysine-deficient APOBEC3G with an N-terminal hemagglutinin (HA) tag fusion (HA-A3G20K/R) was resistant to HIV-1 Vif-induced proteasomal degradation. HA-A3G20K/R molecules were packaged into wild-type HIV-1 particles, and HA-A3G20K/R drastically decreased wild-type HIV-1 reverse transcription products and infectivity. We also showed that the N terminus of A3G was a target of polyubiquitination induced by HIV-1 Vif. Thus, fusion of the HA tag to the N terminus of A3G20K/R reduced its polyubiquitination, the likely mechanism for the resistance of this protein to HIV-1 Vif-induced proteasomal degradation. Finding such ways to induce resistance of A3G to Vif may provide new approaches to anti-HIV/AIDS therapy.

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Section: Discussionmentioning

confidence: 79%

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confidence: 99%

N-Terminal Hemagglutinin Tag Renders Lysine-Deficient APOBEC3G Resistant to HIV-1 Vif-Induced Degradation by Reduced Polyubiquitination

Wang

Shao

et al. 2011

J Virol

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“…The plasmid pAS1B-HA-A3G was constructed previously (8), where the APOBEC3G (A3G) amplified from Homo sapiens cDNA clone IMAGE:1284557, was kindly provided by S. K. Petersen-Mahrt (14,45). The pAS1B-HA-A3G truncation mutants (65 to 132, 102 to 257, and 195 to 384) were constructed by the PCR-based mutagenesis method with the following primers: A3G 65 -KpnI-5=, 5=-CGGGGTACCTATCCTTATGACG TGCCTGACTATGCCAGCCACCCAGAG-3=; A3G 132 -BamH-3=, 5=-CG GGATCCCTGGTAATCTGGG-3=; A3G 102 -Xba-I-5=, 5=-CTAGTCTAGA AGGGATATGGCCACGTTC-3=; A3G 257 -PstI-3=, 5=-AACTGCAGTCAA TGCGGCCTTCAAGGAA-3=; A3G 195 -XbaI-5=, 5=-GGAGATTTCTAGA CACTCGATG-3=; and A3G 384 -BglII-3=, 5=-ATAGATCTATCGATTCAG TTTTCCTGATT-3=.…”

Section: Methodsmentioning

confidence: 99%

The Cellular Antiviral Protein APOBEC3G Interacts with HIV-1 Reverse Transcriptase and Inhibits Its Function during Viral Replication

Wang

Chen

et al. 2012

J Virol

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101

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cThe cytidine deaminase APOBEC3G (A3G) exerts a multifaceted antiviral effect against HIV-1 infection. First, A3G was shown to be able to terminate HIV infection by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription. Also, a number of studies have indicated that A3G inhibits HIV-1 reverse transcription by a non-editingmediated mechanism. However, the mechanism by which A3G directly disrupts HIV-1 reverse transcription is not fully understood. In the present study, by using a cell-based coimmunoprecipitation (Co-IP) assay, we detected the direct interaction between A3G and HIV-1 reverse transcriptase (RT) in produced viruses and in the cotransfected cells. The data also suggested that their interaction did not require viral genomic RNA bridging or other viral proteins. Additionally, a deletion analysis showed that the RT-binding region in A3G was located between amino acids 65 and 132. Overexpression of the RT-binding polypeptide A3G 65-132 was able to disrupt the interaction between wild-type A3G and RT, which consequently attenuated the anti-HIV effect of A3G on reverse transcription. Overall, this paper provides evidence for the physical and functional interaction between A3G and HIV-1 RT and demonstrates that this interaction plays an important role in the action of A3G against HIV-1 reverse transcription. Several host proteins have been identified as intrinsic restriction factors because of their ability to inhibit HIV replication and/or dissemination (2, 31, 41, 52). Among them, the apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G; here referred to as A3G) is the one that restricts HIV-1 replication through more than one mechanism (17,32,37,38,40). In the absence of the HIV-1 viral infectivity factor (Vif), A3G is incorporated into progeny viruses through its interaction with the nucleocapsid (NC) domain of the Gag protein and/or viral RNA (1,15,56,63). Once these progeny viruses initiate new infection, the incorporated A3G will deaminate the cytidine to uridine in the viral minus-strand DNA during reverse transcription, resulting in hypermutation in the provirus. As a result, the HIV-1 proviral DNA will be no longer functional or degrade rapidly (21,32,38,64). Additionally, the mutated proviral DNA may produce defective or truncated viral polypeptides that represent a significant source of major histocompatibility complex class I (MHC-I)-restricted epitopes to activate HIV-1-specific CD8 ϩ cytotoxic T lymphocytes (CTLs) (12).Reverse transcription catalyzed by HIV-1 reverse transcriptase (RT) is a critical step for HIV-1 to establish its replication cycle. In infected cells, RT employs tRNA 3Lys and the polypurine tract as primers and converts the viral genomic RNA into doublestranded viral DNA (23). This process is catalyzed by both the DNA polymerase and RNase H activities of RT (23,49). Interestingly, in addition to the deaminase activity, A3G has also been shown to directly inhibit HIV-1 reverse transcription by a non...

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“…A very recent study has shown that fusion of A3A, a Vif-resistant member of the APOBEC3 family that does not restrict HIV-1 infectivity, to the HIV-1 protein Vpr resulted in the alteration of its virion localization from the matrix to the viral core, which granted Vpr.A3A anti-HIV activity (44). A second very recent study showed that fusion of A3G to a viriontargeting peptide derived from Vpr (R88-A3G) resulted in restriction of HIV-1 infectivity (45). This fusion protein is susceptible to Vif-mediated degradation, and the higher virion incorporation is due to increased targeting to the virion by the remaining intracellular R88-A3G.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of HIV-1 Infection and Replication by Enhancing Viral Incorporation of Innate Anti-HIV-1 Protein A3G

Green

Liu

2009

Journal of Biological Chemistry

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APOBEC3G (A3G) is a cellular protein that has been identified as an innate anti-human immunodeficiency virus type 1 (HIV-1) factor. One of the major functions of HIV-1 virion infectivity protein (Vif) protein is to target A3G for ubiquitination/proteasome-mediated degradation and, as a result, evade the host innate defense mechanism. Thus, we wished to devise a strategy to restore the anti-HIV activity of A3G by actively targeting it into HIV-1 virions and countering HIV-1 Vif-targeted degradation. In the current study we performed a series of proof-of-concept experiments for this strategy using as a delivery vehicle of A3G, a derivate of non-pathogenic Nef mutant Nef7 that is capable of being efficiently incorporated into HIV-1 virions. We demonstrate that the Nef7.A3G fusion protein retains several important properties of Nef7; that is, the higher virion incorporation efficiency, no PAK-2 (p21-activated kinase 2) activation, and no CD4 and major histocompatibility complex I down-regulation. Meanwhile, we show that virion incorporated Nef7.A3G possesses the anti-HIV infectivity function of A3G. Moreover, we show that virus-like particle-mediated inverse fusion delivery of Nef7.A3G into HIV-infected CD4؉ T lymphocytes leads to potent inhibition of HIV-1 replication in these cells. Taken together, these results indicate that Nef7.A3G can effectively restrict HIV infection and replication by restoring the virion incorporation of A3G, even in the presence of Vif.Apolipoprotein mRNA editing enzyme catalytic peptide 3G (APOBEC3G, or A3G) is an innate antiviral cellular protein that dramatically reduces human immunodeficiency virus type 1 (HIV-1) 2 infectivity when incorporated into virions (1, 2). It is a single-stranded DNA deaminase that functions immediately after viral infection, during the first round of reverse transcription, to deaminate cytidine to uracil on the minus strand of the proviral DNA. This results in hypermutations that lead to loss of genetic stability and protein function (3, 4). The key to the A3G anti-HIV function is virion incorporation. When HIV-1 infects a cell, A3G is incorporated into the progeny virions from that cell through interactions with HIV-1 nucleocapsid protein and RNA. When those virions then infect a second cell, A3G renders them non-replicative. However, lentiviruses such as HIV-1 have evolved a mechanism to prevent the antiviral effects of A3G and other members of APOBEC3 family. The HIV-1 virion infectivity protein (Vif) binds to A3G and targets it for degradation by recruiting the E3 ubiquitin ligase cullin 5-elongin B/C (5), thus preventing A3G encapsidation (6 -9).HIV-1 Nef protein is an accessory protein of ϳ27 kDa. It is post-translationally modified by phosphorylation and by the addition of a myristoyl moiety to its second amino acid (glycine), which aids in its membrane targeting and is required for many Nef functions (10 -12). Although Nef is dispensable for in vitro HIV-1 replication, it is essential for efficient HIV-1 replication and pathogenesis in vivo (13-16). ...

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Vpr14-88-Apobec3G Fusion Protein Is Efficiently Incorporated into Vif-Positive HIV-1 Particles and Inhibits Viral Infection

Cited by 32 publications

References 68 publications

N-Terminal Hemagglutinin Tag Renders Lysine-Deficient APOBEC3G Resistant to HIV-1 Vif-Induced Degradation by Reduced Polyubiquitination

N-Terminal Hemagglutinin Tag Renders Lysine-Deficient APOBEC3G Resistant to HIV-1 Vif-Induced Degradation by Reduced Polyubiquitination

The Cellular Antiviral Protein APOBEC3G Interacts with HIV-1 Reverse Transcriptase and Inhibits Its Function during Viral Replication

Inhibition of HIV-1 Infection and Replication by Enhancing Viral Incorporation of Innate Anti-HIV-1 Protein A3G

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