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Archived Comments for: Turning up the volume on mutational pressure: Is more of a good thing always better? (A case study of HIV-1 Vif and APOBEC3)

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  1. Testing a hypothesis with full disclosure

    Harold Smith, University of Rochester

    1 April 2008

    The article by S.K. Pillai J.K. Wong and J.D. Barbour (2008) 'Turning up the volume on mutational pressure: is more of a good thing always better? (a case study on HIV-1 Vif and APOBEC3') Retrovirology 5:26, tests the hypothesis that inhibition of HIV-1 Vif may accelerate the evolution of drug resistance and immune escape due the mutagenic effects A3G and A3F coupled with viral recombination. The authors address the hypothesis using computational methods to search an HIV-1 subtype B database for the occurrence and frequency of mutations that match consensus sequences known to be targeted deaminated by A3G and A3F using the protease sequence as a model HIV open reading frame. The authors find numerous G to A mutations within the protease coding region, some of which correlate with known mutations attributable to protease drug resistant phenotypes. As the authors rightly point out, it will take substantial additional effort and longitudinal studies to determine whether disrupting Vif inhibition of A3G and A3F can drive the emergence of drug resistance strains or provide effective therapy for HIV/AIDS. Their words of caution are appropriate.

    However in writing this manuscript, the authors’ referencing was incomplete and important publications that do not support the hypothesis were not discussed.

    The authors state: "There does not appear to be any correlation between levels of APOBEC3 expression (mRNA) in peripheral blood mononuclear cells and HIV viral loads or CD4+ counts in untreated HIV-infected individuals [43], although a more rigorous study design may be required to adequately address this relationship [44].

    It should be noted that reference 44 described a hypothesis that there is a distribution of A3G expression levels in the human population and that the level of A3G may affect HIV infectivity and AIDS progression. This hypothesis was based on a publication (1) that was omitted from the authors' discussion, and that predated ref 43. In this paper Jin et al. demonstrated that A3G mRNA expression levels were correlated with reduced viremia and increased CD4+ counts. The study's inclusion of samples from long term nonprogressors (LTNP) revealed that A3G mRNA expression in LTNP was among the highest measured. The question of A3G expression levels in healthy and HIV/AIDS patents needs to be further evaluated as both the investigation of Jin et al., and that in of ref. 43 involved a limited number of patients. However, this point but this does not change the fact that the authors are obligated to give fair disclosure to this published data representing a divergent view pointthere is an open question.

    The second inaccurate statement is: "Multiple reports demonstrate that APOBEC3G is catalytically activity and has antiviral potency as a monomer. Hence the monomeric capacity of APOBEC3G makes the range of 3-11 molecules per virion meaningful, and likely consequential [28,29]". This is misleading and over interpreted because the oligomeric state of APOBEC enzymes are unresolved. No one has done definitive work to demonstrate the macromolecular organization of APOBEC3G as it deaminates deoxy cytidine.

    There are two manuscripts that conclude that full length APOBEC3G is a monomer which are ref 29 cited by the authors and the work of Wichroski et al. (3) (which the authors do not reference). The second article cited by the authors (ref 28), describes expression of only a fragment of A3G that contains a highly mutated catalytic domain (see also ref. 2). Although this A3G fragment bound ssDNA as a monomer and had deaminase activity in cells as a GST fusion protein, its oligomeric state during catalysis was not assessed directly.

    On the other hand, there are structural and functional data that suggest that full length and catalytically active A3G may be a dimer (4,5) and that A3G in cells resistant to HIV infection, has a molecular mass equivalent to a dimer (6-8). It is of interest to note that all cytidine deaminases that have been structurally and functionally studied to date have been characterized as dimeric or tetrameric (17). From this vantage alone, it would be remarkable if full length APOBEC3G is catalytically active on ssDNA as a monomer. But in all fairness, there is uncertainty concerning the oligomeric state of all APOBEC proteins.

    Crystal structures of APOBEC2 (9) and a yeast homology that deaminates C to U in RNA (10) and DNA (11) show that these proteins are multimeric. Biochemical studies (12,13) and genetic analyses (11,14) have suggested that APOBEC1 dimers are the functional unit. Biochemical studies suggest that Activation Induced Deaminase (AID) dimers are required for somatic hypermutation (15). Although modeling based on the crystal structure of truncated APOBEC2 suggested that AID could be a dimer (9), atomic force microscopy suggested that AID is a monomeric protein (16). When one understands this complexity, it is not apparent why the authors consider the imprecise number of '3-11' A3G molecules per virion as being "meaningful" relative to the "the monomeric capacity of A3G".

    1. Jin X, Brooks A, Chen H, Bennett R, Reichman R, & Smith H.C. (2005) APOBEC3G/CEM15 (hA3G) mRNA Levels Associate Inversely with Human Immunodeficiency Virus Viremia. J. Virology, 79:11513-6.

    2. Chen KM, et al. (2008) Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G. Nature 452: 116-19.

    3. Wichroski MJ, Robb GB, Rana TM (2006) Human Retroviral Host Restriction Factors APOBEC3G and APOBEC3F Localize to mRNA Processing Bodies. PLoS Pathog 2: e41.

    4. Wedekind JE, et al. (2006) Nanostructures of APOBEC3G support a hierarchical assembly model of high molecular mass ribonucleoprotein particles from dimeric subunits. J Biol Chem 281: 38122-26.

    5. Linda Chelico, Phuong Pham, Peter Calabrese & Myron F Goodman (2006) APOBEC3G DNA deaminase acts processively 3’-5’ on single-stranded DNA. Nature Struct. & Mol. Cell. Biol. 13:392-99.

    6. Stopak, K.S., Chiu, Y.L., Kropp, J., Grant, R.M., and Greene, W.C. (2007) Distinct patterns of cytokine regulation of APOBEC3G expression and activity in primary lymphocytes, macrophages, and dendritic cells. J Biol Chem. 282:3539-46.

    7. Chiu, Y.-L., Soros, V.B., Kreisberg, J.F., Stopak, K., Yonemoto, W. and Greene, W.C. (2005) Cellular APOBEC3G restricts HIV-1 infection in resting CD41 T cells. Nature 435:108-14.

    8. Wang, X., Dolan, P.T., Dang, Y. and Zheng, Y.-H. (2007) Biochemical Differentiation of APOBEC3F and APOBEC3G Proteins Associated with HIV-1 Life Cycle. J. Biol. Chem. 282: 1585–94.

    9. Prochnow, C., Bransteitter, R., Klein, M.G., Goodman, M.F. and Chen, X.S. (2007) The APOBEC-2 crystal structure and functional implications for the deaminase AID. Nature 445:447-51

    10. Kefang X., Sowden, M.P., Dance, G.S.C., Torelli, A.T., Smith, H.C., Wedekind, J.E. (2004) The structure of a Yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1. P.N.A.S. USA 101:8114-19.

    11. Smith. H.C. (2007) Measuring editing activity and identifying C to U mRNA editing factors in cells and biochemical isolates. in Methods in Enzymology, RNA Editing & Modification (Gott, J., ed.) Academic Press, NY. 424:387-416.

    12. A., Bhattacharya, S., Carter, C. and Scott, J. (1998) Escherichia coli Cytidine Deaminase Provides a Molecular Model for ApoB RNA Editing and a Mechanism for RNA Substrate Recognition. J. Mol Biol. 275:695-714.

    13. Lau, P.P., Zhu, H-J., Baldini, A., Charnsangavej, C., and Chan, L. (2004) Dimer structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene. PNAS USA 91:8522-26.

    14. Oka, K., Kobayashi, K., Sullivan, M., Martinez, J., Teng, B.B., Ishimura-Oka, K. and Chan, L. (1997) Tissue-specific inhibition of apolipoprotein B mRNA editing in the liver by adenovirus-mediated transfer of a dominant negative mutant APOBEC-1 leads to increased low density lipoprotein in mice. J Biol Chem. 272: 1456-60.

    15. Wang J, Shinkura R., Muramatsu, M, Nagaoka, H, Kinoshita, K Honjo, T (2006) Identification of a specific domain required for dimerization of activation-induced cytidine deaminase. J Biol Chem 281: 19115-23.

    16. Brar SS, Watson, M, Diaz, M (2008) Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA. DNA Repair (Amst) 7: 77-87.

    17. MacElrevey C, Wedekind JE (2008) In RNA and DNA Editing: Molecular Mechanisms and Their Integration into Biological Systems. (ed Smith HC) Wiley, New York, pp 369-419.

    Harold C. Smith, Ph.D.

    Department of Biochemistry and Biophysics

    Univerisity of Rochester

    601 Elmwood Ave, Box 712

    Rochester 14642, USA

    Phone: 585 275-4267

    Fax: 585 275-6007

    Xia Jin, MD, PhD

    Department of Medicine,

    Infectious Diseases Unit

    University of Rochester

    601 Elmwood Ave Box 689

    Rochester 14642, United States

    Phone: 585 275-6515

    Fax: 585 442-9328

    E-mail: xia_jin@urmc.rochester.edu

    Competing interests

    none

  2. In the interest of full disclosure. . .

    Satish Pillai, University of California, San Francisco

    7 April 2008

    In response to Dr. Smith’s comment, “Testing a hypothesis with full disclosure,” we agree that the references he mentioned should have been included in our discussion of APOBEC3-based interventions. However, to clarify, the paper was not intended to be a comprehensive review of the literature on APOBEC3 biology or associated antiretroviral strategies. Moreover, consideration of these additional references does not appreciably change the overall conclusions of the paper.

    In regards to our statement, “There does not appear to be any correlation between levels of APOBEC3 expression (mRNA) in peripheral blood mononuclear cells and HIV viral loads or CD4+ counts in untreated HIV-infected individuals [1], although a more rigorous study design may be required to adequately address this relationship [2],” Dr. Smith correctly points out that an earlier publication from his laboratory [3] did in fact report that A3G mRNA expression levels were correlated with reduced viremia and increased CD4+ counts. However, we chose to focus on the evidence presented by Cho and colleagues [1] due to critical experimental design differences between these two papers. To quote directly from the discussion in the Cho paper [1]:

    “Jin et al. also analyzed the expression of hA3G in the PBMCs of 25 HIV-infected subjects and found that hA3G expression was higher in HIV-uninfected than in HIV-infected subjects, in agreement with our findings. However, in stark contrast to our study, these authors found statistically significant correlations between hA3G expression and viral load (inverse) as well as CD4 cell count (positive). A plausible explanation for this divergence may be that Jin et al. stimulated PBMCs with anti-CD3 and anti-CD28 antibodies prior to RNA extraction. . . We propose that examining unstimulated PBMCs is more representative of the “physiologic” steady state of T cells in vivo, presumably reflecting the population actively resisting HIV infection and replication. Additionally, our subjects were specifically selected to include only those whose HIV status was at steady state in the absence of antiretroviral therapy.”

    As suggested in the Cho paper, the activation of PBMCs prior to analysis of expression calls into question the interpretability and physiologic relevance of the findings by Jin et al. Nevertheless, as suggested by Jin and colleagues in their Retrovirology hypothesis piece [2], a study involving much larger sample sizes will be necessary before any firm conclusions can be drawn regarding the relationship between APOBEC3 expression and HIV pathogenesis. In addition, an APOBEC3-based intervention may be successful even if no such relationship exists; natural variation in APOBEC3 expression and incorporation may fall below a critical antiviral threshold which can only be achieved through HIV-1 Vif inhibition.

    In regards to our statement “Multiple reports demonstrate that APOBEC3G is catalytically active and has antiviral potency as a monomer. Hence the monomeric capacity of APOBEC3G makes the range of 3-11 molecules per virion meaningful, and likely consequential [4, 5],” we were suggesting that the dynamic range of APOBEC3 incorporation into HIV virions as demonstrated by Xu et al [6] should allow for variation in extent of viral hypermutation, especially if each individual molecule has editing capacity. Xu and colleagues claim, based on their observed stoichiometry of 1:439 molecules of A3G to capsid and previous estimates of 1400–5000 capsid proteins per virion, that there are 7 (+-4) A3G molecules per delta-Vif virion.

    Dr. Smith presents additional evidence supporting the monomeric capacity of APOBEC3G [7], and several references supporting a multimeric model of APOBEC3, including extrapolative observations of other APOBEC family members and cytidine deaminases. Given an approximate range of 3-11 molecules per virion, a catalytically active dimer would still present the opportunity for a gradient. On the other hand, if APOBEC3 only functions as a tetramer, there may be little room for modulation. Once again, it is hard to draw any firm conclusions; in Dr. Smith’s own words, there is uncertainty concerning the oligomeric state of all APOBEC proteins, and no one has done definitive work to demonstrate the macromolecular organization of APOBEC3G as it deaminates deoxycytidine. For now we can only say that some of the structural data do in fact point to a monomeric or dimeric capacity of APOBEC3, and once again, the HIV-1 sequence data strongly support the notion of an editing gradient.

    We appreciate the response from Dr. Smith, and believe the information provided in his statement allows for a more balanced perspective on the ideas presented in our paper.

    -Satish K. Pillai, Ph.D.

    UCSF Department of Medicine / NCIRE

    4150 Clement St. (111W3)

    San Francisco, CA 94121

    Phone: (415) 221-4810 x3318

    Fax: (415) 379-5600

    Email: satish.pillai@ucsf.edu

    Website: www.supersatish.com

    1. Cho SJ, Drechsler H, Burke RC, Arens MQ, Powderly W, Davidson NO: APOBEC3F and APOBEC3G mRNA levels do not correlate with human immunodeficiency virus type 1 plasma viremia or CD4+ T-cell count. J Virol 2006, 80:2069-2072.

    2. Jin X, Wu H, Smith H: APOBEC3G levels predict rates of progression to AIDS. Retrovirology 2007, 4:20.

    3. Jin X, Brooks A, Chen H, Bennett R, Reichman R, Smith H: APOBEC3G/CEM15 (hA3G) mRNA levels associate inversely with human immunodeficiency virus viremia. J Virol 2005, 79:11513-11516.

    4. Chen KM, Martemyanova N, Lu Y, Shindo K, Matsuo H, Harris RS: Extensive mutagenesis experiments corroborate a structural model for the DNA deaminase domain of APOBEC3G. FEBS Lett 2007, 581:4761-4766.

    5. Opi S, Takeuchi H, Kao S, Khan MA, Miyagi E, Goila-Gaur R, Iwatani Y, Levin JG, Strebel K: Monomeric APOBEC3G is catalytically active and has antiviral activity. J Virol 2006, 80:4673-4682.

    6. Xu H, Chertova E, Chen J, Ott DE, Roser JD, Hu WS, Pathak VK: Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions. Virology 2007, 360:247-256.

    7. Wichroski MJ, Robb GB, Rana TM: Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies. PLoS Pathog 2006, 2:e41.

    Competing interests

    No competing interests

  3. Suggestions for further reading: experimental validation of our proposed scenario

    Satish Pillai, University of California, San Francisco

    12 April 2008

    Two very recent papers validate the results of our sequence analysis and evolutionary model:

    The first paper is entitled “Cytidine deamination induced HIV-1 drug resistance” by Mulder et al [1]. Mulder and colleagues demonstrate in vitro that partially defective HIV-1 Vif mutants induce high viral genetic diversity and result in the accumulation of 3TC-resistant proviruses prior to drug exposure. In addition, as suggested in the discussion of our paper, they performed serial passaging of virus in the presence of drug (3TC), demonstrating that recombination between wildtype HIV-1 and hypermutant drug-resistant proviruses accelerates the evolution of drug resistance.

    The second paper is entitled “Sequence editing by Apolipoprotein B RNA-editing catalytic component-B and epidemiological surveillance of transmitted HIV-1 drug resistance.” by Gifford et al [2]. Their analysis of 6437 protease and reverse transcriptase sequences from therapy-naive individuals revealed that nearly 3% of proviral sequences obtained from whole blood and 0.2% of samples obtained from plasma harbor drug resistance mutations that are most likely caused by APOBEC3-mediated editing. Although the authors are primarily concerned with the artifactual inflation of transmitted drug resistance estimates resulting from undetected lethal editing, their results validate and extend our observations that APOBEC3 cytidine deaminase activity is frequently associated with the generation of drug resistance mutations in vivo.

    -Satish K. Pillai, Ph.D.

    UCSF Department of Medicine / NCIRE

    4150 Clement St. (111W3)

    San Francisco, CA 94121

    Phone: (415) 221-4810 x3318

    Fax: (415) 379-5600

    Email: satish.pillai@ucsf.edu

    Website: www.supersatish.com

    1. Mulder LC, Harari A, Simon V: Cytidine deamination induced HIV-1 drug resistance. Proc Natl Acad Sci U S A 2008, 105:5501-5506.

    2. Gifford RJ, Rhee SY, Eriksson N, Liu TF, Kiuchi M, Das AK, Shafer RW: Sequence editing by Apolipoprotein B RNA-editing catalytic component-B and epidemiological surveillance of transmitted HIV-1 drug resistance. Aids 2008, 22:717-725.

    Competing interests

    No competing interests

  4. Mutational pressure in the long run

    Viktor Müller, Eötvös Loránd University

    25 September 2008

    I have read the article with great interest. I see one important factor missing in this, and in other studies of mutational pressure induced by APOBEC3 proteins. There is no doubt that the effect of APOBEC influences the nucleotide composition and mutation patterns of HIV in the short run. However, the long-term effect of this mechanism on the nucleotide composition of HIV and other, potentially susceptible viruses is much less clear. There is a logical link between G->A mutational pressure and the adenine-rich genome of HIV and other retroviruses, as stated also in this paper: "The overall highly adenine-biased base composition and codon usage of HIV-1 may in fact reflect adaptation to APOBEC-mediated mutational pressure". However, that link has hardly been tested. One possible way to test such claims is to compare the genomes of multiple viruses that might be susceptible to APOBEC, to test for the expected genomic patterns. In a comparative analysis of a large number of viruses (Müller, V. & Bonhoeffer, S. (2005) Guanine–adenine bias: a general property of retroid viruses that is unrelated to host-induced hypermutation. Trends Genet, 21, 264-268.), we found multiple lines of evidence indicating that the adenine-rich genome of many retroviruses is probably independent of APOBEC-induced mutational pressure. For example, G->A bias in the third letter of degenerate codons showed very strong correlation with C->T bias among retroviruses. Thus, the mechanism that is responsible for the bias must act on both nucleotide strands with comparable efficiency -- which is not the case for the effect of the APOBEC family. Furthermore, the molecular signature of the APOBEC effect (e.g. preference to mutate G's followed by G or A) was not apparent in the viruses that had an overall G->A bias. Thus, while APOBEC-induced mutational pressure indeed shifts the abundance of certain types of point mutations, this effect must be counteracted by some selection force over long-term evolution.

    Competing interests

    There is no competing interest.

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