- Open Access
Vaccinia virus replication is not affected by APOBEC3 family members
© Kremer et al; licensee BioMed Central Ltd. 2006
- Received: 30 August 2006
- Accepted: 19 October 2006
- Published: 19 October 2006
The APOBEC3G protein represents a novel innate defense mechanism against retroviral infection. It facilitates the deamination of the cytosine residues in the single stranded cDNA intermediate during early steps of retroviral infection. Most poxvirus genomes are relatively A/T-rich, which may indicate APOBEC3G-induced mutational pressure. In addition, poxviruses replicate exclusively in the cytoplasm where APOBEC3G is located. It was therefore tempting to analyze whether vaccinia virus replication is affected by APOBEC3G.
The replication of vaccinia virus, a prototype poxvirus, was not, however, inhibited in APOBEC3G-expressing cells, nor did other members of the APOBEC3 family alter vaccinia virus replication. HIV counteracts APOBEC3G by inducing its degradation. However, Western blot analysis showed that the levels of APOBEC3G protein were not affected by vaccinia virus infection.
The data indicate that APOBEC3G is not a restriction factor for vaccinia virus replication nor is vaccinia virus able to degrade APOBEC3G.
- Human Immunodeficiency Virus
- RK13 Cell
- APOBEC3 Protein
- Vector Titer
- APOBEC3 Family
During evolution, eukaryotic cells had to cope with a large amount of pathogens. The interaction of host and pathogen required defense responses in the host, which resulted in the development of the innate immune system. Due to this high selection pressure, pathogens developed strategies to escape or manipulate the host immune defense. Poxviridae in particular, evolved several mechanisms for immune evasion [1, 2]. The best known members of this family are variola and vaccinia virus. Variola virus is the causative agent of smallpox, and although eradicated in the early 70s, it still represents a serious threat as a possible agent for bioterrorism. Vaccinia virus (VACV) is the prototype poxvirus and is frequently used as a vector for vaccine development. The large double-stranded DNA genome of poxviruses is 130 to 300 kb in size, and although most genomes are completely sequenced, the function of many genes necessary for viral infection, replication and immune evasion are not known .
APOBEC3G is a recently discovered defense mechanism against retroviral infection . The protein becomes encapsidated into retroviral particles and is transported into the infected cell, where it facilitates deamination of cytosine residues in the single stranded cDNA intermediate during early steps of infection. APOBEC3G has been shown to be an exclusive DNA mutator . The replacement of C with U in the DNA minus strand during reverse transcription leads to G to A transitions in the plus strand. APOBEC3G, therefore, triggers G to A hypermutations in the newly synthesized viral DNA. The inhibition of viral replication is due either to degradation of the cDNA by the DNA repair machinery or to the lethality of the hypermutations.
In addition to its anti-retroviral function [4, 6], APOBEC3G is also able to restrict hepadnaviruses , and its gene family member APOBEC3A inhibits parvovirus replication . APOBEC3G is located in the cytoplasm of the cell where it performs its function. VACV undergoes its complete viral life cycle in the cytoplasm, and most poxvirus genomes are relatively A/T-rich, which could be caused by APOBEC3G-induced mutational pressure . We were, therefore, interested to determine whether APOBEC3G is also a restricting factor for this virus.
Poxviruses are large cytoplasmic DNA viruses and infection of a cell initiates drastic responses that aim to eliminate virus-infected cells. This innate immune response is able to restrict a multitude of viruses by various strategies. The APOBEC3 gene family contains recently discovered factors that are able to restrict retroviruses, hepadna viruses and parvoviruses [16, 7, 8]. The recently described inhibition of a parvovirus by human APOBEC3A shows that APOBEC proteins also target DNA viruses that replicate in the nucleus without passing through an RNA intermediate . The G and F members of the human APOBEC3 family have two highly conserved zinc-binding domains, characteristic of the catalytic domain (CD) of all cytidine deaminases, and trigger G-to-A hypermutations in the newly synthesized viral DNA. The APOBEC3H cytidine deaminases domain, however, is evolutionary distinct . In contrast to primates, rodents encode only one APOBEC3 protein. This protein cannot inhibit the murine leukemia virus (MLV) but, like APOBEC3G and -F, it is able to restrict HIV-1 [18–20]. Human APOBEC3H is poorly expressed and has no apparent antiretroviral activity .
APOBEC3G, -F and -H, and mouse APOBEC3 are located in the cytoplasm of the cell, the location of poxviral replication. Most poxvirus genomes are relatively A/T-rich which could be a consequence of APOBEC3-induced mutational pressure . It was, therefore, of interest to analyze whether VACV replication is affected by APOBEC3G. However, we could show that APOBEC3G, -F or -H, or mouse APOBEC3 expression has no effect on VACV replication. A limitation of our experiments might be the lack of a positive control, showing that APOBEC3 still confers an inhibitory function on retroviruses in the context of a VACV infection. However, the experiment is not doable. VACV infection leads to a shut down of cellular protein synthesis, which also inhibits retroviral particle formation. But we were able to show the inhibitory effect of APOBEC3G on retroviral and lentiviral vectors using the same experimental setup which did not result in an inhibition of VACV replication, confirming the significance of the study.
VACV has a very broad host range in vitro and is able to infect virtually all cell types . Surprisingly, it has been shown recently that VACV tropism in hematopoietic cells is very restricted. Only poor infection of T lymphocytes, which express APOBEC3G, has been observed . However, activated T-cells are permissive to VACV and our data support the concept that a receptor that permits VACV entry is missing from resting T cells .
Poxviruses have developed several strategies to evade the innate immune system . Like HIV, which encodes Vif that induces degradation of APOBEC3G, VACV could encode a protein to overcome APOBEC3G. We investigated the protein levels of APOBEC3G during VACV infection and our results show that infection does not lead to a degradation of the protein. However, it cannot be excluded that VACV has evolved another mechanism to escape inhibition by APOBEC3G.
Using transient transfections, we could show that APOBEC3G, -F or -H, or mouse APOBEC3 expression has no effect on VACV replication and VACV infection does not lead to a degradation of the APOBEC3G protein.
Plasmids and transfections
The following plasmids were used for transfections: pcDNA-APOBEC3G-MycHis encoding a C-terminally Myc-tagged human APOBEC3G , human APOBEC3F or -H , or mouse APOBEC3  and the empty expression vector pRC-CMV. Retroviral vectors were generated by transfection of the plasmid pHIT60, encoding the MLV Gag/Pol region ; pEnv wt(HX), encoding the ecotropic MLV envelope protein  and pSFG-EGFP, a MLV-based retroviral vector encoding GFP . Transfection of pHIT60, pEnv wt (HX) and pSFG-EGFP into 293T cell results in the production of infectious vector particles, which are able to transduce the GFP encoding vector sequences into target cells. Lentiviral vectors were generated by transient transfections using the following plasmids: pRRLsinCMV-GFPpre, pMDLg/pRRE, pRSVrev and a VSV-G envelope glycoprotein expression plasmid .
Cell culture, transfections viral transduction and determination of titers
293T and NIH 3T3 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; Cambrex, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS); (GIBCO/BRL, Eggenstein, Germany). HeLa, HeLa-APOBEC3G and BHK cells were grown in Roswell Park Memorial Institute-1640 Medium (RPMI-1640; Cambrex, Verviers, Belgium) supplemented with 10% FCS. RK-13 cells were grown in Eagle's Minimum Essential Medium (EMEM; Cambrex, Verviers, Belgium) supplemented with 10% FCS and 1% Non-Essential Amino Acids (Biochrom AG, Berlin, Germany).
To generate MLV-based vector particles, a day before transfection, cells were seeded at a density of 2 × 106 cells in a 10 cm tissue culture plate. The cells were transfected with 2 μg Gag/Pol expression plasmid (pHIT60) , 1 μg ecotropic MLV Env expression plasmid (pEnv ) and 3 μg GFP encoding vector plasmid  using the Fugene reagent (Roche, Penzberg, Germany). Either 2 μg APOBEC3G expression plasmid or the empty vector pRC-CMV was added in addition to assess the effect of APOBEC3G on vector titers. After two days of culture, serial dilutions of viral supernatants from transfected 293T cells were passed through 0.45-μm filters (Greiner, Frickenhausen, Germany) and incubated with 2 × 105 NIH 3T3 cells. 48–72 hours after transduction, the numbers of GFP-expressing cells were detected by FACS analysis. The titers are given in relative infectious units and are representative data of three independent experiments.
Lentiviral vectors were generated by seeding 293T cells at a density of 2 × 106 cells in a 10 cm tissue culture plate one day before transfection. The cells were transfected with the following expression plasmids  using the Fugene reagent (Roche, Penzberg): pRRLsinCMV-GFPpre, pMDLg/pRRE, pRSVrev and a VSV-G envelope glycoprotein expression plasmid. 48 hrs after transfection the vector supernatants were harvested, filtered and used for transductions. Titers were determined as described above for retroviral vectors.
For the analysis of VACV replication, cells (BHK or 293T) were transfected with 8 μg plasmid DNA, encoding APOBEC3 proteins and infected with vaccinia virus WR 48 h after the transfection. Cells were harvested at the indicated time points and vaccinia virus was titrated on RK-13 cells.
Transfection of APOBEC3G expression plasmid was analyzed by intracellular immunofluorescence staining with a mouse anti-Myc antibody (BD-Biosciences, Heidelberg, Germany) and a FITC-conjugated anti-mouse antibody after permeabilization of the cells with BD Perm/Wash Buffer (BD Pharmingen, Heidelberg, Germany). The numbers of FITC-stained cells were detected by FACS analysis.
Western blot analysis
Cell lysates were obtained and Western blot analysis was performed as described previously . Western blot analysis was performed with the following antibodies: mouse anti-6xHis antibody (Acris, Hiddenhausen, Germany), mouse anti-β-actin antibody (Sigma-Aldrich, Munich, Germany), polyclonal rabbit antiserum directed against the vaccinia virus E3 protein (kind gift of B. Jacobs) and horseradish peroxidase-coupled sheep anti-mouse IgG antibodies or protein A (Amersham Biosciences, Freiburg, Germany). Detection was performed using an enhanced chemiluminescence Western blot detection kit (Amersham Biosciences, Freiburg, Germany).
The work was supported by the Deutsche Forschungsgemeinschaft (GK 1172). We are grateful to D. Kabat for kindly providing the plasmid pcDNA-APOBEC3G-Myc and Catherine Haynes for critically reading the manuscript.
- Seet BT, Johnston JB, Brunetti CR, Barrett JW, Everett H, Cameron C, Sypula J, Nazarian SH, Lucas A, McFadden G: Poxviruses and immune evasion. Annu Rev Immunol 2003, 21: 377-423. 10.1146/annurev.immunol.21.120601.141049View ArticlePubMedGoogle Scholar
- Haga IR, Bowie AG: Evasion of innate immunity by vaccinia virus. Parasitology 2005, 130 Suppl: S11-S25. 10.1017/S0031182005008127View ArticlePubMedGoogle Scholar
- Moss B: Vaccinia virus: a tool for research and vaccine development. Science 1991, 252: 1662-1667. 10.1126/science.2047875View ArticlePubMedGoogle Scholar
- Cullen BR: Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J Virol 2006, 80: 1067-1076. 10.1128/JVI.80.3.1067-1076.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Suspene R, Sommer P, Henry M, Ferris S, Guetard D, Pochet S, Chester A, Navaratnam N, Wain-Hobson S, Vartanian JP: APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase. Nucl Acids Res 2004, 32: 2421-2429. 10.1093/nar/gkh554PubMed CentralView ArticlePubMedGoogle Scholar
- Kremer M, Schnierle BS: HIV-1 Vif: HIV's weapon against the cellular defense factor APOBEC3G. Curr HIV Res 2005, 3: 339-344. 10.2174/157016205774370456View ArticlePubMedGoogle Scholar
- Turelli P, Mangeat B, Jost S, Vianin S, Trono D: Inhibition of hepatitis B virus replication by APOBEC3G. Science 2004, 303: 1829. 10.1126/science.1092066View ArticlePubMedGoogle Scholar
- Chen H, Lilley CE, Yu Q, Lee DV, Chou J, Narvaiza I, Landau NR, Weitzman MD: APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons. Curr Biol 2006, 16: 480-485. 10.1016/j.cub.2006.01.031View ArticlePubMedGoogle Scholar
- Muller V, Bonhoeffer S: Guanine-adenine bias: a general property of retroid viruses that is unrelated to host-induced hypermutation. Trends Genet 2005, 21: 264-268. 10.1016/j.tig.2005.03.004View ArticlePubMedGoogle Scholar
- Marin M, Rose KM, Kozak SL, Kabat D: HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat Med 2003, 9: 1398-403. 10.1038/nm946View ArticlePubMedGoogle Scholar
- Soneoka Y, Cannon PM, Ramsdale EE, Griffiths JC, Romano G, Kingsman SM, Kingsman AJ: A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res 1995, 23: 628-33.PubMed CentralView ArticlePubMedGoogle Scholar
- Erlwein O, Wels W, Schnierle BS: Chimeric ecotropic MLV envelope proteins that carry EGF receptor-specific ligands and the Pseudomonas exotoxin A translocation domain to target gene transfer to human cancer cells. Virology 2002, 302: 333-41. 10.1006/viro.2002.1517View ArticlePubMedGoogle Scholar
- Lindemann D, Bock M, Schweizer M, Rethwilm A: Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins. J Virol 1997, 71: 4815-20.PubMed CentralPubMedGoogle Scholar
- Sheehy AM, Gaddis NC, Malim MH: The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat Med 2003, 9: 1404-7. 10.1038/nm945View ArticlePubMedGoogle Scholar
- Conticello SG, Harris RS, Neuberger MS: The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G. Curr Biol 2003, 13: 2009-13. 10.1016/j.cub.2003.10.034View ArticlePubMedGoogle Scholar
- Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, Trono D: Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 2003, 424: 99-103. 10.1038/nature01709View ArticlePubMedGoogle Scholar
- Conticello SG, Thomas CJ, Petersen-Mahrt SK, Neuberger MS: Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. Mol Biol Evol 2005, 22: 367-377. 10.1093/molbev/msi026View ArticlePubMedGoogle Scholar
- Langlois MA, Beale RC, Conticello SG, Neuberger MS: Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities. Nucleic Acids Res 2005, 33: 1913-1923. 10.1093/nar/gki343PubMed CentralView ArticlePubMedGoogle Scholar
- Douaisi M, Dussart S, Courcoul M, Bessou G, Vigne R, Decroly E: HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles. Biochem Biophys Res Commun 2004, 321: 566-573. 10.1016/j.bbrc.2004.07.005View ArticlePubMedGoogle Scholar
- Kremer M, Bittner A, Schnierle BS: Human APOBEC3G incorporation into murine leukemia virus particles. Virology 2005, 337: 175-182. 10.1016/j.virol.2005.04.006View ArticlePubMedGoogle Scholar
- OhAinle M, Kerns JA, Malik HS, Emerman M: Adaptive evolution and antiviral activity of the conserved mammalian cytidine deaminase APOBEC3H. J Virol 2006, 80: 3853-3862. 10.1128/JVI.80.8.3853-3862.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Moss B: Poxvirus entry and membrane fusion. Virology 2006, 344: 48-54. 10.1016/j.virol.2005.09.037View ArticlePubMedGoogle Scholar
- Sanchez-Puig JM, Sanchez L, Roy G, Blasco R: Susceptibility of different leukocyte cell types to Vaccinia virus infection. Virol J 2004, 1: 10. 10.1186/1743-422X-1-10PubMed CentralView ArticlePubMedGoogle Scholar
- Chahroudi A, Chavan R, Koyzr N, Waller EK, Silvestri G, Feinberg MB: Vaccinia virus tropism for primary hematolymphoid cells is determined by restricted expression of a unique virus receptor. J Virol 2005, 79: 10397-10407. 10.1128/JVI.79.16.10397-10407.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Muckenfuss H, Hamdorf M, Held U, Perkovic M, Lower J, Cichutek K, Flory E, Schumann GG, Munk C: APOBEC3 proteins inhibit human LINE-1 retrotransposition. J Biol Chem 2006, 281: 22161-22172. 10.1074/jbc.M601716200View ArticlePubMedGoogle Scholar
- Mariani R, Chen D, Schrofelbauer B, Navarro F, Konig R, Bollman B, Munk C, Nymark-McMahon H, Landau NR: Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 2003, 114: 21-31. 10.1016/S0092-8674(03)00515-4View ArticlePubMedGoogle Scholar
- Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L: A third-generation lentivirus vector with a conditional packaging system. J Virol 1998, 72: 8463-8471.PubMed CentralPubMedGoogle Scholar
- Schnierle BS, Moritz D, Jeschke M, Groner B: Expression of chimeric envelope proteins in helper cell lines and integration into Moloney murine leukemia virus particles. Gene Ther 1996, 3: 334-42.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.