- Email: [email protected]
HIV-1 Vif versus APOBEC3G: newly appreciated warriors in the ancient battle between virus and host
Update
TRENDS in Microbiology
To understand how the disappearance of ubiquitinylated proteins relates to toxin sensitivity, it must be established whether there is a direct link between MEK inactivation and proteasomal pathway activity or whether an unidentified LeTx-responsive pathway leads to this event. The report by Salles et al. [23] demonstrates clear molecular differences between toxin-sensitive and toxinresistant cell types. Using these differences as a starting point, the mechanism of TIR must now be elucidated. If QTLs do control toxin sensitivity (Kif1c, Ltxs2 and Ltxs3), understanding how this influences MEK– ERK activity, ubiquitinylated protein stability and TIR will probably provide insights into the cellular response to LF activity. Questions still remain, including whether or not TIR is associated with reduced cellular levels of cytokines, because previous studies demonstrate that sub-lethal doses of toxin downregulate cytokine production [12,13]. It should be noted that TIR cells might be a tool to study novel ERK activation mechanisms, therefore having implications beyond the scope of anthrax pathogenesis. Finally, it must now be determined if TIR is relevant in vivo and, if so, how TIR applies to anthrax pathogenesis and host immunity. References 1 Metchnikoff, E. (1905) Immunity in Infective Diseases, Cambridge University Press 2 Scobie, H.M. et al. (2003) Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc. Natl. Acad. Sci. U. S. A. 100, 5170 – 5174 3 Bradley, K.A. et al. (2001) Identification of the cellular receptor for anthrax toxin. Nature 414, 225– 229 4 Smith, H. and Keppie, J. (1954) Observations on experimental anthrax: demonstration of a specific lethal factor produced in vivo by Bacillus anthracis. Nature 173, 869– 870 5 Smith, H. and Stanley, J.L. (1962) Purification of the third factor of anthrax toxin. J. Gen. Microbiol. 29, 517– 521 6 O’Brien, J. et al. (1985) Effects of anthrax toxin components on human neutrophils. Infect. Immun. 47, 306 – 310 7 Friedlander, A.M. (1986) Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J. Biol. Chem. 261, 7123 – 7126 8 Smith, H. et al. (1955) The chemical basis of the virulence of bacillus
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anthracis. IV: Secondary shock as the major factor in death of guineapigs from anthrax. Br. J. Exp. Pathol. 36, 323 – 335 Hanna, P.C. et al. (1993) On the role of macrophages in anthrax. Proc. Natl. Acad. Sci. U. S. A. 90, 10198 – 10201 Park, J.M. et al. (2002) Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 297, 2048– 2051 Kim, S.O. et al. (2003) Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factoralpha. J. Biol. Chem. 278, 7413– 7421 Erwin, J.L. et al. (2001) Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin. Infect. Immun. 69, 1175 – 1177 Pellizzari, R. et al. (1999) Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett. 462, 199 – 204 Agrawal, A. et al. (2003) Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin. Nature 424, 329– 334 Watters, J.W. et al. (2001) Kif1C, a kinesin-like motor protein, mediates mouse macrophage resistance to anthrax lethal factor. Curr. Biol. 11, 1503 – 1511 McAllister, R.D. et al. (2003) Susceptibility to anthrax lethal toxin is controlled by three linked quantitative trait Loci. Am. J. Pathol. 163, 1735– 1741 Tang, G. and Leppla, S.H. (1999) Proteasome activity is required for anthrax lethal toxin to kill macrophages. Infect. Immun. 67, 3055– 3060 Tucker, A.E. et al. (2003) Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages. Cell. Microbiol. 5, 523 – 532 Bhatnagar, R. and Friedlander, A.M. (1994) Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity. Infect. Immun. 62, 2958 – 2962 Bhatnagar, R. et al. (1989) Calcium is required for the expression of anthrax lethal toxin activity in the macrophage-like cell line J774A.1. Infect. Immun. 57, 2107 – 2114 Bhatnagar, R. et al. (1999) Activation of phospholipase C and protein kinase C is required for expression of anthrax lethal toxin cytotoxicity in J774A.1 cells. Cell. Signal. 11, 111 – 116 Kau, J.H. et al. (2002) Calyculin A sensitive protein phosphatase is required for Bacillus anthracis lethal toxin induced cytotoxicity. Curr. Microbiol. 44, 106 – 111 Salles, I.I. et al. (2003) Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages. Proc. Natl. Acad. Sci. U. S. A. 100, 12426 – 12431
0966-842X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.02.003
HIV-1 Vif versus APOBEC3G: newly appreciated warriors in the ancient battle between virus and host Elias G. Argyris and Roger J. Pomerantz The Dorrance H. Hamilton Laboratories, Center for Human Virology and Biodefense, Division of Infectious Diseases and Environmental Medicine, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Suite 329, Philadelphia, Pennsylvania 19107, USA
Recent studies demonstrate that apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), the newly identified target of HIV-1 Vif, represents an endogenous inhibitor of HIV-1 Corresponding author: Roger J. Pomerantz ([email protected]). www.sciencedirect.com
replication and is a viral-encapsidated cellular protein that deaminates minus-strand reverse transcript cytosine residues to uracils. HIV-1 Vif counteracts the inhibitory activity of APOBEC3G by forming a complex with the enzyme, inducing its degradation and preventing its viral encapsidation. This finding provides
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valuable insights into virus–host interactions and suggests a potential, novel anti-HIV-1 therapeutic approach. For nearly two decades the molecular mechanisms involved in the mode of action and function of the HIV-1 Vif accessory protein have remained a mystery. The recent identification and characterization of the target of Vif and its main function in blocking the action of the host antiviral protein APOBEC3G, as reported by several independent investigators, have greatly contributed to a major breakthrough and took us a giant leap forward in exploring one of the ‘final frontiers’ in HIV-1 molecular virology [1 – 5]. The role of Vif in viral replication The vif gene was originally discovered in the mid 1980s and was initially called sor [6,7]. The gene product is a 23 kDa basic protein encoded by all lentiviruses, except for equine infectious anemia virus, and has been renamed Vif (virion infectivity factor). In contrast to other HIV-1 accessory proteins (Nef, Vpr, Vpu and Vpx), Vif is unique in its phenotype, both qualitatively and quantitatively. Earlier studies demonstrated that a Vif-mutant HIV-1 (Dvif HIV-1) does not replicate in primary human T-cells, macrophages or certain CD4þ-transformed T-cell lines (known as ‘non-permissive’), whereas other transformed T-cell lines, such as 293, HeLa, SupT1 and CEM-SS, known as ‘permissive’, support Dvif HIV-1 replication [7 – 9]. Interestingly, Vif-mutant HIV-1 virus derived from permissive cells can infect non-permissive cells in a single round of infection, but the Dvif HIV-1 derived from these non-permissive cells has reduced infectivity compared with wild-type virus (i.e. Vif-intact) and fails to complete a single cycle of replication. Over the past decade this enigma remained mechanistically unsolved; why do Dvif HIV-1 virions produced by non-permissive cells show no differences in protein, RNA composition or reverse transcriptase activity from their counterparts derived from permissive cells, and why are they able to initiate reverse transcription but fail to complete double-stranded cDNA synthesis and reach the proviral stage? Studies have shown that Vif binds to HIV-1 RNA, however, the mechanisms by which Dvif HIV-1 is generated remain unclear [10– 12]. A major clue to the solution of this mystery came in 1998 by two independent groups, Madani and Kabat [13] and Simon et al. [14]. In somatic cell fusion experiments, heterokaryons formed by the fusion of permissive and non-permissive cells were found to be non-permissive. This finding suggested that nonpermissiveness is caused by an endogenous cellular factor, an inhibitor of HIV-1, which was overcome by the viral Vif protein. Soon after, Sheehy et al. [15] discovered the answer. Using a novel polymerase chain reaction (PCR)based cDNA subtraction strategy, the cellular factor was identified; initially named CEM15, this cellular factor was found to specifically inhibit the replication of Dvif HIV-1. CEM15 was found to be identical to the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G). This enzyme is a member of the cytidine deaminase family of nucleic acid-editing enzymes, with an www.sciencedirect.com
Vol.12 No.4 April 2004
unknown physiological role in normal cells. As a result of its activity, 1 – 2% of all the cytosine residues in the viral DNA are converted to uracil. The mutations are observed both in pre- and post-integrated proviral HIV-1 DNA, and there is a sequence-specificity with a preference for the third dC residue in a Py-C-C sequence [2– 3,5]. But the mechanisms of interaction between APOBEC3G and HIV-1 virions, Vif or other viral proteins, its mode of action and the possibility of functional differences between APOBEC3G proteins from different species, still remained open issues. Antiviral activity of APOBEC3G Recent reports on the identification and characterization of HIV-1 Vif ’s target, APOBEC3G, shed further light on the field and greatly improved our understanding of viral – cell interactions [1 –5] (Figure 1). Mariani et al. [1] for the first time demonstrated that the antiviral activity of APOBEC3G was maintained across diverse species in spite of extensive amino acid sequence differences and regardless of whether lentiviruses infect the species or not. Their findings showed that mouse, macaque and African green monkey APOBEC3Gs are potent inhibitors of wildtype and Dvif HIV-1. Similar to the human enzyme, the non-homologous APOBEC3Gs were virion-encapsidated and induced cytosine deamination to uracil in the viral minus-strand reverse transcripts. Vif could efficiently block virion encapsidation of human APOBEC3G, however, it failed to prevent the encapsidation of nonhomologous APOBEC3G, which was present in virions at high levels regardless of Vif. They demonstrated that HIV-1 Vif can form a complex with human but not mouse APOBEC3G, suggesting that Vif-resistant inhibition is probably caused by a failure of HIV-1 Vif to interact with non-homologous APOBEC3G and block its encapsidation. As shown in Figure 1, in non-permissive cells infected with Dvif HIV-1, APOBEC3G is packaged into the virions during viral assembly. When APOBEC3G-containing Dvif virions infect a new cell, minus-strand cDNA reverse transcription is initiated and the encapsidated APOBEC3G deaminates cytosine residues in the newly synthesized DNA. The incorporated uracils might destabilize the DNA, causing its degradation by DNA base excision repair enzymes. In a non-permissive cell infected with wild-type HIV-1, Vif binds to APOBEC3G preventing its encapsidation, therefore rescuing the minus-strand cytosines from deamination and subsequent DNA degradation. In cells expressing a nonhomologous APOBEC3G, Vif fails to prevent APOBEC3G encapsidation. Minus-strand cytosine residues are deaminated resulting in uracil-containing reverse transcripts, which are poor substrates for integration. The preference for minus-strand degradation could be caused by a requirement to deaminate singlestranded DNA that is generated by the RNaseH activity of reverse transcriptase. Vif blocks APOBEC3G via a proteasome-dependent pathway The precise mechanism by which Vif blocks APOBEC3G is not clear. Recent studies by Stopak et al. [16] demonstrated that Vif prevents virion incorporation of endogenous
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(b) Mutant HIV-1 virion (∆Vif)
(a) Wild-type HIV-1 (Vif intact) virion
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TRENDS in Microbiology
Figure 1. APOBEC3G–HIV-1 Vif interactions and their effects on HIV-1 replication in non-permissive cells. (a) In cells infected with the wild-type HIV-1 (intact Vif), Vif binds to the cellular factor APOBEC3G and induces its degradation, therefore preventing its encapsidation in virions and resulting in productive viral replication. (b) In cells infected with mutant HIV-1 (DVif), APOBEC3G is packaged with the viral genome in HIV-1 particles. Upon infection of a new cell and during reverse transcription, APOBEC3G deaminates 20 -deoxycytidines (c) in the DNA minus (2 ) strand, producing 20 -deoxyuridines (U). Plus-strand synthesis converts the C ! U changes into G ! A mutations. These hypermutations could lead to strand breakage, destruction and impairment of subsequent viral functions.
APOBEC3G by effectively depleting the intracellular levels of this enzyme in HIV-1-infected T-cells. Their findings suggest that Vif achieves this depletion by impairing the translation of APOBEC3G mRNA and accelerating the posttranslational degradation of the enzyme by the 26S proteasome [16]. The proteasome involvement in APOBEC3G degradation upon interaction with Vif was also confirmed by Sheehy et al. [17], whereas Marin et al. [18] have further suggested that the most conserved sequence in Vif, an SLQ(Y/F)LA motif, although unnecessary for binding to APOBEC3G is required for the degradation of the enzyme by a proteasome-dependent pathway. Moreover, the findings of Mariani et al. [1] suggest that HIV-1 Vif can recognize, bind and therefore inactivate human APOBEC3G in a specific manner. Murine and African green monkey enzymes are also efficiently targeted for incorporation into HIV-1 virions assembled in these hosts; they possess strong anti-HIV-1 activities and can induce hypermutation effects similar to human APOBEC3G. Interestingly, murine, Chinese hamster, and the majority of the primate APOBEC3Gs are relatively resistant to the effects of HIV-1 Vif and can therefore inhibit even wild-type HIV-1 replication [1]. Altogether these findings suggest that HIV-1 Vif has evolved specifically to recognize and inhibit human APOBEC3G, allowing effective viral replication in humans. www.sciencedirect.com
Another interesting finding is that these enzymes are incorporated into virions of other retroviruses, such as murine leukemia virus (MLV). Harris et al. [3] and Zhang et al. [2] have recently reported HIV-1 Vif-sensitive inhibition of MLV by human APOBEC3G. MLV has a yet unidentified mechanism of resisting APOBEC3G-mediated deamination, as it lacks a Vif analogue. Therefore, MLV can replicate in the presence of enzymatically active APOBEC3G, which is expressed in mouse leukocytes and encapsidated into MLV virions [1]. Conclusions The newly identified HIV-1 Vif target, and the direct or indirect interactions of APOBEC3G with Vif, provide valuable insights into the complex virus – host interactions, and more importantly suggest potential, novel anti-HIV-1 therapeutic approaches. These might include the development and use of potent non-peptide Vif inhibitors, in the form of peptidomimetics or small organic molecular inhibitors [16 – 20]. In addition, recent studies have provided strong evidence for the existence of other important cellular factors, such as tyrosine kinase, Hck and a lymphocyte-specific nuclear body protein, Sp140, which have been suggested to have functional roles in inhibiting HIV-1 replication, the effects of which can be
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counteracted by HIV-1 Vif [21,22]. It is possible that Vif might be fully functional and active in interactions that involve multiple targets. Cells have evolved an astonishing number of mechanisms to protect themselves from viral invaders, however, viruses have developed stealthy mechanisms to escape these inhibitory pathways. The battle between cells and retroviruses is an ancient and on-going one. APOBEC3G, Hck and Sp140 are not the only recently identified cellular defenders in this battle. Gao et al. [23] have reported on ZAP, a protein that targets and destroys viral mRNAs, and Hatziioannou et al. [24] have described the antiretroviral factors Fv1 and Lv1, which target the capsid proteins of incoming retroviral cores of the viral DNA. Though overall we should not expect these new findings to translate to antiviral treatment for patients in the near future, they certainly bring us a giant step ahead and make biomedical scientists more hopeful in the battle against pathogenic viral infections. References 1 Mariani, R. et al. (2003) Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114, 21– 31 2 Zhang, H. et al. (2003) The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424, 94– 98 3 Harris, R.S. et al. (2003) DNA deamination mediates innate immunity to retroviral infection. Cell 113, 803– 809 4 Lecossier, D. et al. (2003) Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science 300, 1112 5 Mangeat, B. et al. (2003) Broad antiretroviral defense by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424, 99– 103 6 Fisher, A.G. et al. (1987) The sor gene of HIV-1 is required for efficient virus transmission in vitro. Science 237, 888 – 893 7 Strebel, K. et al. (1987) The HIV ‘A’ (sor) gene product is essential for virus infectivity. Nature 328, 728– 773 8 Gabuzda, D.H. et al. (1992) Role of Vif in replication of human immunodeficiency virus type 1 in CD4 þ T lymphocytes. J. Virol. 66, 6489 – 6495 9 von Schwedler, U. et al. (1993) Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells. J. Virol. 67, 4945 – 4955
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10 Zhang, H. et al. (2000) Human immunodeficiency virus type 1 Vif protein is an integral component of an mRNP complex of viral RNA and could be involved in the viral RNA folding and packaging process. J. Virol. 74, 8252– 8261 11 Khan, M.A. et al. (2000) Human immunodeficiency virus type 1 Vif protein is packaged into the nucleoprotein complex through an interaction with viral genomic RNA. J. Virol. 76, 8252– 8261 12 Dettenhofer, M. et al. (2000) Association of human immunodeficiency virus type 1 Vif with RNA and its role in reverse transcription. J. Virol. 74, 8938 – 8945 13 Madani, N. and Kabat, D. (1998) An endogenous inhibitor of human immunodeficiency virus in human lymphocytes is overcome by the viral Vif protein. J. Virol. 72, 10251 – 10255 14 Simon, J.H.M. et al. (1998) Evidence for a newly discovered cellular anti-HIV-1 phenotype. Nat. Med. 4, 1397 – 1400 15 Sheehy, A.M. et al. (2002) Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418, 646– 650 16 Stopak, K. et al. (2003) HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability. Mol. Cell 12, 591 – 601 17 Sheehy, A.M. et al. (2003) The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat. Med. 9, 1404– 1407 18 Marin, M. et al. (2003) HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat. Med. 9, 1398 – 1403 19 Yang, S. et al. (2001) The multimerization of human immunodeficiency virus type 1 (HIV-1) Vif protein: a requirement for Vif function in the viral life-cycle. J. Biol. Chem. 276, 4889– 4893 20 Yang, B. et al. (2003) Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins. J. Biol. Chem. 278, 6596– 6602 21 Hassaine, G. et al. (2001) The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral Vif protein. J. Biol. Chem. 276, 16885 – 16893 22 Madani, N. et al. (2002) Implication of the lymphocyte-specific nuclear body protein Sp140 in an innate response to human immunodeficiency virus type 1. J. Virol. 76, 11133– 11138 23 Gao, G. et al. (2002) Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein. Science 297, 1703 – 1706 24 Hatziioannou, T. et al. (2003) Restriction of multiple divergent retroviruses by Lv1 and Ref1. EMBO J. 22, 385 – 394
0966-842X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.02.004
| Genome Analysis
Gene duplication and biased functional retention of paralogs in bacterial genomes Dirk Gevers, Klaas Vandepoele, Cedric Simillion and Yves Van de Peer Bioinformatics and Evolutionary Genomics, Ghent University/Flanders Interuniversity Institute for Biotechnology (VIB), Technologiepark 927, B-9052 Ghent, Belgium
Gene duplication is considered an important prerequisite for gene innovation that can facilitate adaptation to changing environments. The analysis of 106 bacterial genome sequences has revealed the existence of a significant number of paralogs. Analysis of the functional classification of these paralogs reveals a preferential enrichment in functional classes that are involved in Corresponding author: Yves Van de Peer ([email protected]). www.sciencedirect.com
transcription, metabolism and defense mechanisms. From the organization of paralogs in the genome we can conclude that duplicated genes in bacteria appear to have been mainly created by small-scale duplication events, such as tandem and operon duplications. Microbial genomes have a considerable fraction of genes that are homologous to other genes within the same genome [1,2]. There are basically two ways through which
TRENDS in Microbiology
To understand how the disappearance of ubiquitinylated proteins relates to toxin sensitivity, it must be established whether there is a direct link between MEK inactivation and proteasomal pathway activity or whether an unidentified LeTx-responsive pathway leads to this event. The report by Salles et al. [23] demonstrates clear molecular differences between toxin-sensitive and toxinresistant cell types. Using these differences as a starting point, the mechanism of TIR must now be elucidated. If QTLs do control toxin sensitivity (Kif1c, Ltxs2 and Ltxs3), understanding how this influences MEK– ERK activity, ubiquitinylated protein stability and TIR will probably provide insights into the cellular response to LF activity. Questions still remain, including whether or not TIR is associated with reduced cellular levels of cytokines, because previous studies demonstrate that sub-lethal doses of toxin downregulate cytokine production [12,13]. It should be noted that TIR cells might be a tool to study novel ERK activation mechanisms, therefore having implications beyond the scope of anthrax pathogenesis. Finally, it must now be determined if TIR is relevant in vivo and, if so, how TIR applies to anthrax pathogenesis and host immunity. References 1 Metchnikoff, E. (1905) Immunity in Infective Diseases, Cambridge University Press 2 Scobie, H.M. et al. (2003) Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc. Natl. Acad. Sci. U. S. A. 100, 5170 – 5174 3 Bradley, K.A. et al. (2001) Identification of the cellular receptor for anthrax toxin. Nature 414, 225– 229 4 Smith, H. and Keppie, J. (1954) Observations on experimental anthrax: demonstration of a specific lethal factor produced in vivo by Bacillus anthracis. Nature 173, 869– 870 5 Smith, H. and Stanley, J.L. (1962) Purification of the third factor of anthrax toxin. J. Gen. Microbiol. 29, 517– 521 6 O’Brien, J. et al. (1985) Effects of anthrax toxin components on human neutrophils. Infect. Immun. 47, 306 – 310 7 Friedlander, A.M. (1986) Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J. Biol. Chem. 261, 7123 – 7126 8 Smith, H. et al. (1955) The chemical basis of the virulence of bacillus
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anthracis. IV: Secondary shock as the major factor in death of guineapigs from anthrax. Br. J. Exp. Pathol. 36, 323 – 335 Hanna, P.C. et al. (1993) On the role of macrophages in anthrax. Proc. Natl. Acad. Sci. U. S. A. 90, 10198 – 10201 Park, J.M. et al. (2002) Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 297, 2048– 2051 Kim, S.O. et al. (2003) Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factoralpha. J. Biol. Chem. 278, 7413– 7421 Erwin, J.L. et al. (2001) Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin. Infect. Immun. 69, 1175 – 1177 Pellizzari, R. et al. (1999) Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett. 462, 199 – 204 Agrawal, A. et al. (2003) Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin. Nature 424, 329– 334 Watters, J.W. et al. (2001) Kif1C, a kinesin-like motor protein, mediates mouse macrophage resistance to anthrax lethal factor. Curr. Biol. 11, 1503 – 1511 McAllister, R.D. et al. (2003) Susceptibility to anthrax lethal toxin is controlled by three linked quantitative trait Loci. Am. J. Pathol. 163, 1735– 1741 Tang, G. and Leppla, S.H. (1999) Proteasome activity is required for anthrax lethal toxin to kill macrophages. Infect. Immun. 67, 3055– 3060 Tucker, A.E. et al. (2003) Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages. Cell. Microbiol. 5, 523 – 532 Bhatnagar, R. and Friedlander, A.M. (1994) Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity. Infect. Immun. 62, 2958 – 2962 Bhatnagar, R. et al. (1989) Calcium is required for the expression of anthrax lethal toxin activity in the macrophage-like cell line J774A.1. Infect. Immun. 57, 2107 – 2114 Bhatnagar, R. et al. (1999) Activation of phospholipase C and protein kinase C is required for expression of anthrax lethal toxin cytotoxicity in J774A.1 cells. Cell. Signal. 11, 111 – 116 Kau, J.H. et al. (2002) Calyculin A sensitive protein phosphatase is required for Bacillus anthracis lethal toxin induced cytotoxicity. Curr. Microbiol. 44, 106 – 111 Salles, I.I. et al. (2003) Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages. Proc. Natl. Acad. Sci. U. S. A. 100, 12426 – 12431
0966-842X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.02.003
HIV-1 Vif versus APOBEC3G: newly appreciated warriors in the ancient battle between virus and host Elias G. Argyris and Roger J. Pomerantz The Dorrance H. Hamilton Laboratories, Center for Human Virology and Biodefense, Division of Infectious Diseases and Environmental Medicine, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Suite 329, Philadelphia, Pennsylvania 19107, USA
Recent studies demonstrate that apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), the newly identified target of HIV-1 Vif, represents an endogenous inhibitor of HIV-1 Corresponding author: Roger J. Pomerantz ([email protected]). www.sciencedirect.com
replication and is a viral-encapsidated cellular protein that deaminates minus-strand reverse transcript cytosine residues to uracils. HIV-1 Vif counteracts the inhibitory activity of APOBEC3G by forming a complex with the enzyme, inducing its degradation and preventing its viral encapsidation. This finding provides
146
Update
TRENDS in Microbiology
valuable insights into virus–host interactions and suggests a potential, novel anti-HIV-1 therapeutic approach. For nearly two decades the molecular mechanisms involved in the mode of action and function of the HIV-1 Vif accessory protein have remained a mystery. The recent identification and characterization of the target of Vif and its main function in blocking the action of the host antiviral protein APOBEC3G, as reported by several independent investigators, have greatly contributed to a major breakthrough and took us a giant leap forward in exploring one of the ‘final frontiers’ in HIV-1 molecular virology [1 – 5]. The role of Vif in viral replication The vif gene was originally discovered in the mid 1980s and was initially called sor [6,7]. The gene product is a 23 kDa basic protein encoded by all lentiviruses, except for equine infectious anemia virus, and has been renamed Vif (virion infectivity factor). In contrast to other HIV-1 accessory proteins (Nef, Vpr, Vpu and Vpx), Vif is unique in its phenotype, both qualitatively and quantitatively. Earlier studies demonstrated that a Vif-mutant HIV-1 (Dvif HIV-1) does not replicate in primary human T-cells, macrophages or certain CD4þ-transformed T-cell lines (known as ‘non-permissive’), whereas other transformed T-cell lines, such as 293, HeLa, SupT1 and CEM-SS, known as ‘permissive’, support Dvif HIV-1 replication [7 – 9]. Interestingly, Vif-mutant HIV-1 virus derived from permissive cells can infect non-permissive cells in a single round of infection, but the Dvif HIV-1 derived from these non-permissive cells has reduced infectivity compared with wild-type virus (i.e. Vif-intact) and fails to complete a single cycle of replication. Over the past decade this enigma remained mechanistically unsolved; why do Dvif HIV-1 virions produced by non-permissive cells show no differences in protein, RNA composition or reverse transcriptase activity from their counterparts derived from permissive cells, and why are they able to initiate reverse transcription but fail to complete double-stranded cDNA synthesis and reach the proviral stage? Studies have shown that Vif binds to HIV-1 RNA, however, the mechanisms by which Dvif HIV-1 is generated remain unclear [10– 12]. A major clue to the solution of this mystery came in 1998 by two independent groups, Madani and Kabat [13] and Simon et al. [14]. In somatic cell fusion experiments, heterokaryons formed by the fusion of permissive and non-permissive cells were found to be non-permissive. This finding suggested that nonpermissiveness is caused by an endogenous cellular factor, an inhibitor of HIV-1, which was overcome by the viral Vif protein. Soon after, Sheehy et al. [15] discovered the answer. Using a novel polymerase chain reaction (PCR)based cDNA subtraction strategy, the cellular factor was identified; initially named CEM15, this cellular factor was found to specifically inhibit the replication of Dvif HIV-1. CEM15 was found to be identical to the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G). This enzyme is a member of the cytidine deaminase family of nucleic acid-editing enzymes, with an www.sciencedirect.com
Vol.12 No.4 April 2004
unknown physiological role in normal cells. As a result of its activity, 1 – 2% of all the cytosine residues in the viral DNA are converted to uracil. The mutations are observed both in pre- and post-integrated proviral HIV-1 DNA, and there is a sequence-specificity with a preference for the third dC residue in a Py-C-C sequence [2– 3,5]. But the mechanisms of interaction between APOBEC3G and HIV-1 virions, Vif or other viral proteins, its mode of action and the possibility of functional differences between APOBEC3G proteins from different species, still remained open issues. Antiviral activity of APOBEC3G Recent reports on the identification and characterization of HIV-1 Vif ’s target, APOBEC3G, shed further light on the field and greatly improved our understanding of viral – cell interactions [1 –5] (Figure 1). Mariani et al. [1] for the first time demonstrated that the antiviral activity of APOBEC3G was maintained across diverse species in spite of extensive amino acid sequence differences and regardless of whether lentiviruses infect the species or not. Their findings showed that mouse, macaque and African green monkey APOBEC3Gs are potent inhibitors of wildtype and Dvif HIV-1. Similar to the human enzyme, the non-homologous APOBEC3Gs were virion-encapsidated and induced cytosine deamination to uracil in the viral minus-strand reverse transcripts. Vif could efficiently block virion encapsidation of human APOBEC3G, however, it failed to prevent the encapsidation of nonhomologous APOBEC3G, which was present in virions at high levels regardless of Vif. They demonstrated that HIV-1 Vif can form a complex with human but not mouse APOBEC3G, suggesting that Vif-resistant inhibition is probably caused by a failure of HIV-1 Vif to interact with non-homologous APOBEC3G and block its encapsidation. As shown in Figure 1, in non-permissive cells infected with Dvif HIV-1, APOBEC3G is packaged into the virions during viral assembly. When APOBEC3G-containing Dvif virions infect a new cell, minus-strand cDNA reverse transcription is initiated and the encapsidated APOBEC3G deaminates cytosine residues in the newly synthesized DNA. The incorporated uracils might destabilize the DNA, causing its degradation by DNA base excision repair enzymes. In a non-permissive cell infected with wild-type HIV-1, Vif binds to APOBEC3G preventing its encapsidation, therefore rescuing the minus-strand cytosines from deamination and subsequent DNA degradation. In cells expressing a nonhomologous APOBEC3G, Vif fails to prevent APOBEC3G encapsidation. Minus-strand cytosine residues are deaminated resulting in uracil-containing reverse transcripts, which are poor substrates for integration. The preference for minus-strand degradation could be caused by a requirement to deaminate singlestranded DNA that is generated by the RNaseH activity of reverse transcriptase. Vif blocks APOBEC3G via a proteasome-dependent pathway The precise mechanism by which Vif blocks APOBEC3G is not clear. Recent studies by Stopak et al. [16] demonstrated that Vif prevents virion incorporation of endogenous
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(b) Mutant HIV-1 virion (∆Vif)
(a) Wild-type HIV-1 (Vif intact) virion
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Figure 1. APOBEC3G–HIV-1 Vif interactions and their effects on HIV-1 replication in non-permissive cells. (a) In cells infected with the wild-type HIV-1 (intact Vif), Vif binds to the cellular factor APOBEC3G and induces its degradation, therefore preventing its encapsidation in virions and resulting in productive viral replication. (b) In cells infected with mutant HIV-1 (DVif), APOBEC3G is packaged with the viral genome in HIV-1 particles. Upon infection of a new cell and during reverse transcription, APOBEC3G deaminates 20 -deoxycytidines (c) in the DNA minus (2 ) strand, producing 20 -deoxyuridines (U). Plus-strand synthesis converts the C ! U changes into G ! A mutations. These hypermutations could lead to strand breakage, destruction and impairment of subsequent viral functions.
APOBEC3G by effectively depleting the intracellular levels of this enzyme in HIV-1-infected T-cells. Their findings suggest that Vif achieves this depletion by impairing the translation of APOBEC3G mRNA and accelerating the posttranslational degradation of the enzyme by the 26S proteasome [16]. The proteasome involvement in APOBEC3G degradation upon interaction with Vif was also confirmed by Sheehy et al. [17], whereas Marin et al. [18] have further suggested that the most conserved sequence in Vif, an SLQ(Y/F)LA motif, although unnecessary for binding to APOBEC3G is required for the degradation of the enzyme by a proteasome-dependent pathway. Moreover, the findings of Mariani et al. [1] suggest that HIV-1 Vif can recognize, bind and therefore inactivate human APOBEC3G in a specific manner. Murine and African green monkey enzymes are also efficiently targeted for incorporation into HIV-1 virions assembled in these hosts; they possess strong anti-HIV-1 activities and can induce hypermutation effects similar to human APOBEC3G. Interestingly, murine, Chinese hamster, and the majority of the primate APOBEC3Gs are relatively resistant to the effects of HIV-1 Vif and can therefore inhibit even wild-type HIV-1 replication [1]. Altogether these findings suggest that HIV-1 Vif has evolved specifically to recognize and inhibit human APOBEC3G, allowing effective viral replication in humans. www.sciencedirect.com
Another interesting finding is that these enzymes are incorporated into virions of other retroviruses, such as murine leukemia virus (MLV). Harris et al. [3] and Zhang et al. [2] have recently reported HIV-1 Vif-sensitive inhibition of MLV by human APOBEC3G. MLV has a yet unidentified mechanism of resisting APOBEC3G-mediated deamination, as it lacks a Vif analogue. Therefore, MLV can replicate in the presence of enzymatically active APOBEC3G, which is expressed in mouse leukocytes and encapsidated into MLV virions [1]. Conclusions The newly identified HIV-1 Vif target, and the direct or indirect interactions of APOBEC3G with Vif, provide valuable insights into the complex virus – host interactions, and more importantly suggest potential, novel anti-HIV-1 therapeutic approaches. These might include the development and use of potent non-peptide Vif inhibitors, in the form of peptidomimetics or small organic molecular inhibitors [16 – 20]. In addition, recent studies have provided strong evidence for the existence of other important cellular factors, such as tyrosine kinase, Hck and a lymphocyte-specific nuclear body protein, Sp140, which have been suggested to have functional roles in inhibiting HIV-1 replication, the effects of which can be
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counteracted by HIV-1 Vif [21,22]. It is possible that Vif might be fully functional and active in interactions that involve multiple targets. Cells have evolved an astonishing number of mechanisms to protect themselves from viral invaders, however, viruses have developed stealthy mechanisms to escape these inhibitory pathways. The battle between cells and retroviruses is an ancient and on-going one. APOBEC3G, Hck and Sp140 are not the only recently identified cellular defenders in this battle. Gao et al. [23] have reported on ZAP, a protein that targets and destroys viral mRNAs, and Hatziioannou et al. [24] have described the antiretroviral factors Fv1 and Lv1, which target the capsid proteins of incoming retroviral cores of the viral DNA. Though overall we should not expect these new findings to translate to antiviral treatment for patients in the near future, they certainly bring us a giant step ahead and make biomedical scientists more hopeful in the battle against pathogenic viral infections. References 1 Mariani, R. et al. (2003) Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114, 21– 31 2 Zhang, H. et al. (2003) The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424, 94– 98 3 Harris, R.S. et al. (2003) DNA deamination mediates innate immunity to retroviral infection. Cell 113, 803– 809 4 Lecossier, D. et al. (2003) Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science 300, 1112 5 Mangeat, B. et al. (2003) Broad antiretroviral defense by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424, 99– 103 6 Fisher, A.G. et al. (1987) The sor gene of HIV-1 is required for efficient virus transmission in vitro. Science 237, 888 – 893 7 Strebel, K. et al. (1987) The HIV ‘A’ (sor) gene product is essential for virus infectivity. Nature 328, 728– 773 8 Gabuzda, D.H. et al. (1992) Role of Vif in replication of human immunodeficiency virus type 1 in CD4 þ T lymphocytes. J. Virol. 66, 6489 – 6495 9 von Schwedler, U. et al. (1993) Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells. J. Virol. 67, 4945 – 4955
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10 Zhang, H. et al. (2000) Human immunodeficiency virus type 1 Vif protein is an integral component of an mRNP complex of viral RNA and could be involved in the viral RNA folding and packaging process. J. Virol. 74, 8252– 8261 11 Khan, M.A. et al. (2000) Human immunodeficiency virus type 1 Vif protein is packaged into the nucleoprotein complex through an interaction with viral genomic RNA. J. Virol. 76, 8252– 8261 12 Dettenhofer, M. et al. (2000) Association of human immunodeficiency virus type 1 Vif with RNA and its role in reverse transcription. J. Virol. 74, 8938 – 8945 13 Madani, N. and Kabat, D. (1998) An endogenous inhibitor of human immunodeficiency virus in human lymphocytes is overcome by the viral Vif protein. J. Virol. 72, 10251 – 10255 14 Simon, J.H.M. et al. (1998) Evidence for a newly discovered cellular anti-HIV-1 phenotype. Nat. Med. 4, 1397 – 1400 15 Sheehy, A.M. et al. (2002) Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418, 646– 650 16 Stopak, K. et al. (2003) HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability. Mol. Cell 12, 591 – 601 17 Sheehy, A.M. et al. (2003) The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat. Med. 9, 1404– 1407 18 Marin, M. et al. (2003) HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat. Med. 9, 1398 – 1403 19 Yang, S. et al. (2001) The multimerization of human immunodeficiency virus type 1 (HIV-1) Vif protein: a requirement for Vif function in the viral life-cycle. J. Biol. Chem. 276, 4889– 4893 20 Yang, B. et al. (2003) Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins. J. Biol. Chem. 278, 6596– 6602 21 Hassaine, G. et al. (2001) The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral Vif protein. J. Biol. Chem. 276, 16885 – 16893 22 Madani, N. et al. (2002) Implication of the lymphocyte-specific nuclear body protein Sp140 in an innate response to human immunodeficiency virus type 1. J. Virol. 76, 11133– 11138 23 Gao, G. et al. (2002) Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein. Science 297, 1703 – 1706 24 Hatziioannou, T. et al. (2003) Restriction of multiple divergent retroviruses by Lv1 and Ref1. EMBO J. 22, 385 – 394
0966-842X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.02.004
| Genome Analysis
Gene duplication and biased functional retention of paralogs in bacterial genomes Dirk Gevers, Klaas Vandepoele, Cedric Simillion and Yves Van de Peer Bioinformatics and Evolutionary Genomics, Ghent University/Flanders Interuniversity Institute for Biotechnology (VIB), Technologiepark 927, B-9052 Ghent, Belgium
Gene duplication is considered an important prerequisite for gene innovation that can facilitate adaptation to changing environments. The analysis of 106 bacterial genome sequences has revealed the existence of a significant number of paralogs. Analysis of the functional classification of these paralogs reveals a preferential enrichment in functional classes that are involved in Corresponding author: Yves Van de Peer ([email protected]). www.sciencedirect.com
transcription, metabolism and defense mechanisms. From the organization of paralogs in the genome we can conclude that duplicated genes in bacteria appear to have been mainly created by small-scale duplication events, such as tandem and operon duplications. Microbial genomes have a considerable fraction of genes that are homologous to other genes within the same genome [1,2]. There are basically two ways through which