Miyagy et al JVI revised 1 Stably Expressed APOBEC3F Has Negligible Antiviral Activity 1 Eri ...

41 Pages·2010·2.11 MB·English

JVI Accepts, published online ahead of print on 11 August 2010
J. Virol. doi:10.1128/JVI.01249-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Miyagy et al JVI revised 1
1 Stably Expressed APOBEC3F Has Negligible Antiviral Activity
2 Eri Miyagi 1, Charles R. Brown 1, Sandrine Opi 2, Mohammad Khan 3, Ritu Goila-Gaur 4, Sandra
3 Kao 1, Robert C. Walker Jr. 1, Vanessa Hirsch 1, and Klaus Strebel 1*)
4 1 Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,
5 NIH, Bethesda, Maryland, 20892-0460; 2 current address: Center for Cancer Research, CRCM
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6 U891 Inserm, Marseille, France; 3 current address: Institute for Human Virology, Baltimore, o
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7 MD; 4 current address: Institute of Liver and Biliary Sciences, New Delhi, India lo
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10 Abstract: 190 words h
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13 Running title: APOBEC3F lacks antiviral activity o
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20 *) corresponding author
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22 Correspondent footnote: Klaus Strebel, Viral Biochemistry Section, Laboratory of Molecular
23 Microbiology, NIAID, NIH; Bldg. 4, Room 310; 4 Center Drive MSC 0460; Bethesda, MD
24 20892-0460, Tel. [phone]; Fax. [phone]; E-Mail: [email protected]
Miyagy et al JVI revised 2
1 ABSTRACT
2 APOBEC3F (A3F) is a member of the family of cytidine deaminases that is often coexpressed
3 with APOBEC3G (A3G) in cells susceptible to HIV infection. A3F has been shown to have
4 strong antiviral activity in transient expression studies and together with A3G it is considered the
5 most potent cytidine deaminase targeting HIV. Previous analyses suggested that the antiviral
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6 properties of A3F can be dissociated from its catalytic deaminase activity. We were able to o
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7 confirm the deaminase-independent antiviral activity of exogenously expressed A3F; however, lo
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8 we also noted that exogenous expression was associated with very high A3F mRNA and protein e
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9 levels. In analogy to our previous study on A3G we produced stable HeLa cell lines o
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10 constitutively expressing wild type or deaminase defective A3F at levels that were more in line h
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11 with the levels of endogenous A3F in H9 cells. A3F expressed in stable HeLa cells was //
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12 packaged into Vif-deficient viral particles with similar efficiency as A3G and was properly s
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13 targeted to the viral nucleoprotein complex. Surprisingly, however, neither wild type nor o
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14 deaminase defective A3F inhibited HIV-1 infectivity. These results imply that the antiviral o
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15 activity of endogenous A3F is negligible when compared to A3G. p
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Miyagy et al JVI revised 3
1 INTRODUCTION
2 The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif plays an
3 important role in regulating virus infectivity. It is now well established that Vif counteracts the
4 antiviral activity of several human cytidine deaminases, incl. APOBEC3G (A3G), APOBEC3F
5 (A3F), and APOBEC3DE (reviewed in (10)). However, in spite of Vif, hypermutated HIV-1
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6 sequences have been identified in more than 40% of HIV-infected individuals (17). The o
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7 accumulation of G to A hypermutation was first attributed to an error prone HIV reverse lo
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8 transcriptase (31, 47). However, the identification of APOBEC3G as a major HIV host e
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9 restriction factor targeting single stranded viral DNA leading to G to A hypermutation on the o
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10 plus-strand DNA has put the spotlight on cellular cytidine deaminases as possible contributing h
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11 factors to HIV hypermutation. It is interesting to note that in patient samples G to A //
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12 hypermutations were observed in a preferred GG and GA context and that their appearance was s
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13 independent of the normal accumulation of random mutations (17). It is also interesting that o
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14 hypermutation in the GA dinucleotide context exceeded that in the GG context not only in HIV-1 o
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15 sequences from infected human patients (17) but also in macaques experimentally infected with p
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16 a SHIV variant expressing a mutated Vif (43). Subsequent reports identified matching 5
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17 dinucleotide preferences for A3G (GG) and A3F (GA) (4, 13, 18, 52, 56) arguing for a role of 1
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18 these cytidine deaminases in HIV hypermutation. Such a role of host deaminases is further y

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19 supported by the fact that A3G and A3F are expressed in a wide variety of cell types including e
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20 cells susceptible to HIV infection (4, 23, 28, 52).
21 Transient expression studies have demonstrated that A3F potently inhibits HIV-1
22 replication in a Vif-sensitive manner (4, 8, 28, 52, 55, 58); overall, however, A3F appears to be
23 less sensitive to HIV-1 Vif than A3G (8, 28, 48, 55). Interestingly, whereas dose-response
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1 studies indicated that wt A3G had a stronger inhibitory effect on viral infectivity than its
2 deaminase-defective variant, wt A3F and deaminase-defective A3F inhibited viral infectivity
3 equally well (14). This observation implies that A3F-mediated inhibition of viral infectivity
4 occurs through a predominantly deamination-independent mechanism. Indeed, the existence of a
5 deamination-independent mechanism to inhibit viral infectivity has been widely reported for
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6 A3G and A3F (3, 7, 11, 12, 14, 16, 27, 29, 32-34, 36, 38, 53). Deaminase-independent inhibition o
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7 by A3G was also reported for other viruses such as HTLV-1 and hepatitis B virus (26, 37, 40, lo
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8 42). However, most of these studies were done under conditions of experimentally elevated e
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9 levels of A3G or A3F. Indeed, we and others have found that A3G-dependent inhibition of HIV- o
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10 1 as well as inhibition of the yeast retrotransposon Ty1 and the murine endogenous retrovirus h
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11 MusD require catalytic deaminase activity when A3G expression approaches endogenous levels //
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12 (6, 34, 44). s
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13 Our current study is aimed at further investigating the functional importance of A3F o
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14 catalytic activity for the inhibition of HIV-1 replication. We employed a strategy similar to the o
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15 one used for the analysis of A3G (34). First, we performed a titration of exogenously expressed p
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16 wild type and deaminase-defective A3F. Consistent with published reports we found that in such 5
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17 a setting A3F had strong antiviral activity but that deaminase activity was not important for the 1
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18 inhibition of HIV-1 infectivity. We next established stable HeLa cell lines expressing wild type y

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19 or deaminase-defective A3F. We found that virus produced from these cells incorporated A3F e
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20 with similar efficiency as A3G. However, inhibition of A3F packaging by Vif was less efficient
21 than A3G consistent with the reported relative insensitivity of A3F to Vif. Surprisingly, neither
22 wild type nor deaminase-deficient A3F produced in the stable HeLa lines inhibited HIV-1
23 infectivity and hypermutation of viral genomes was not detected. In conclusion, our data suggest
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1 that (i) A3F is not a major contributor to deaminase-dependent inhibition of viral infectivity and
2 (ii) the deaminase-independent inhibition of viral infectivity is largely restricted to assay systems
3 involving transient overexpression of A3F.
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Miyagy et al JVI revised 6
1 MATERIALS AND METHODS
2 Plasmids.
3 The full-length molecular clone of HIV-1 (pNL4-3) was used for the production of wild
4 type infectious virus (1). Construction of its vif-defective variant pNL4-3Vif(-) was described
5 previously (21). A vector for the expression of myc-tagged A3G wt in the backbone of
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6 pcDNA3.1(-) (Invitrogen Corp., Carlsbad, CA) has been previously described (19). Vector o
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7 pcDNA3.1-APOBEC3F-V5-6XHis was obtained from Matija Peterlin and Yong-Hui Zheng lo
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8 through the NIH AIDS Research and Reference Reagent Program (Cat #10100). To replace the e
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9 V5 tag by a C-terminal myc tag, the A3F insert was sub-cloned into pcDNA3.1(-)MycHis using o
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10 standard molecular biological techniques. Mutation of cysteine residues C280 and C283 in h
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11 human A3F in the backbone of A3F-myc was accomplished by PCR-directed mutagenesis. The //
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12 presence of the desired mutations was verified by sequence analysis. as
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14 Cell culture, transfections, and construction of HeLa-A3F cell lines. o
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15 HeLa cells were propagated in Dulbecco’s modified Eagles medium (DMEM) containing p
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16 10% fetal bovine serum. For transient transfection of HeLa cells, cells were grown in 25 cm2 5
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17 flasks to about 80% confluence. Cells were transfected using TransIT-LT1 (Mirus Corp., 1
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18 Madison WI) following the manufacturer’s recommendations. Unless noted otherwise, a total of y

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19 5 µg of plasmid DNA per 25 cm2 flask (~5x 106 cells) was used. Cells were harvested 24 to 48 h e
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20 post-transfection. HeLa cell lines stably expressing C-terminally Myc-tagged wild type A3F
21 (A3F wt), deaminase-defective A3F C280S/C283A (A3F mt), wild type A3G (A3G wt), or
22 containing empty vector (Ctrl) were constructed by transfecting HeLa cells in a 10 cm dish with
23 pcDNA-A3F-myc, pcDNA-A3F-myc C280S/C283A, pcDNA-APO3G-Myc, or empty
Miyagy et al JVI revised 7
1 pcDNA3.1(-), respectively. Two days following transfection, cells were treated with G418 (800
2 µg/ml) and cultured until G418 resistant colonies became apparent. Individual colonies were
3 transferred to 24 well plates and maintained in DMEM containing 10% fetal bovine serum and
4 400 µg/ml of G418. Cells were passaged three times and then screened by immunoblot analysis
5 for A3F expression using a myc-specific antibody. Two clones each of wild type (A3F wt) and
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6 deaminase defective A3F C280S/C283A (A3F mt) and one clone each of wild type A3G (A3G o
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7 wt) and empty vector (Ctrl) were selected for functional studies. lo
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9 Monocytes derived macrophage and IFN alpha treatment. o
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10 Elutriated monocytes from healthy donors were allowed to differentiate into macrophages in 6- t
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11 well plates (4 x 106 per well) in 2 ml of complete DMEM containing10 % pooled human serum /jv
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12 (PHS) for 7 days (35). The cells were then treated with IFN alpha 2c (0 to 10 ng/ml) for 24 h and s
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13 analyzed for protein expression and mRNA levels by imunoblotting or northern blot analysis, r
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16 Antibodies. ,

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17 A peptide antibody to human A3G was prepared by immunizing rabbits with KLH- 1
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18 coupled peptides corresponding to residues 367 to 384 of human A3G (20). A monoclonal y

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19 antibody to Vif (MAb #319) and a polyclonal antibody to A3F were obtained from Michael e
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20 Malim through the NIH AIDS Research and Reference Reagent Program (Cat #6459 & #11474)
21 (14, 45). A monoclonal antibody to c-myc (Sigma-Aldrich, Inc., St. Louis MO) was used for
22 detection of epitope tagged A3F and A3G. For identification of tubulin and actin, commercial
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1 monoclonal antibodies were used (Sigma-Aldrich, Inc., St. Louis MO). An HIV-positive patient
2 serum was used for the identification of HIV-1 capsid (CA) and matrix (MA) proteins.
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4 Immunocytochemistry.
5 Cells were grown in LabTek 8 well chambers (Nalge Nunc International, IL) overnight and fixed
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6 with 1% paraformaldehyde. The samples were rinsed in phosphate buffered saline (PBS), o
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7 blocked with methanol/hydrogen peroxide, rinsed and blocked with PBS and normal goat serum. lo
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8 The primary antibody, rabbit polyclonal APO3F (pre absorbed with HeLa cells at 4˚C, e
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9 overnight) was diluted in PBS and applied to the samples at 1:200 for 1hr followed by DAKO o
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10 EnVision goat anti-rabbit-IgG horse radish peroxidase (HRP) labeled polymer (DAKO, K4002). h
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11 The HRP label was reacted with diaminobenzidine, rinsed and counter stained with hematoxylin. //
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12 For negative control we omitted primary antibody. The labeled samples were viewed and s
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13 photographed with a Zeiss Axio Imager Z1. o
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14 o
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15 Deamination assay. p
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16 For the analysis of virus-associated deaminase activity, virus-containing supernatants (7 5
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17 ml) were pre-cleared by filtration (0.45 µm) and pelleted through 20% sucrose (4 ml). 1
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18 Concentrated virions were directly suspended in 100 µl of deaminase buffer (40 mM Tris, pH y

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19 8.0, 40 mM KCl, 50 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 2% [vol/vol] glycerol, and e
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20 0.1% [vol/vol] Triton X-100) and incubated with a [32P]-labeled oligonucleotide substrate (5’
21 ATTATTATTA TTATACCCAA TTCTTTATTT ATTTATTTAT TT 3’) containing A3G and
22 A3F target sites (underlined). After overnight incubation at 37°C with occasional agitation, the
23 oligonucleotides were purified on G25 Quick Spin columns (Roche) and reacted with uracil-
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1 DNA glycosylase (Roche) for 2 h at 37°C in uracil-DNA glycosylase buffer (60 mM Tris-HCl,
2 pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.001% bovine serum albumin) to remove the uracil
3 bases generated by deamination. To cleave at the abasic site, the samples were treated for 5 min
4 at 37°C with 0.15 M NaOH. Samples were then neutralized by the addition of 0.15 M HCl. The
5 cleaved products were separated on 15% acrylamide-7 M urea gels and detected by
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6 autoradiography. o
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8 Immunoblotting. e
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9 For immunoblot analysis of intracellular proteins, whole cell lysates were prepared as o
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10 follows: Cells were washed once with PBS, suspended in PBS and mixed with an equal volume t
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11 of sample buffer (4% sodium dodecyl sulfate, 125 mM Tris-HCl, pH 6.8, 10% 2- /jv
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12 mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue). To analyze virus-associated s
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13 proteins, cell-free filtered supernatants from transfected HeLa cells (3.5 ml) were pelleted (75
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14 min, 35,000 rpm) through a 20% sucrose cushion (5.5 ml) in an SW41 rotor. The concentrated o
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15 virus pellet was suspended in PBS and mixed with an equal volume of sample buffer. Proteins p
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16 were solubilized by heating 10 to 15 min at 95oC. Cell and virus lysates were subjected to SDS- 5,

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17 PAGE; proteins were transferred to PVDF membranes and reacted with appropriate antibodies as 1
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18 described in the text. Membranes were then incubated with horseradish peroxidase-conjugated y

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19 secondary antibodies (Amersham Biosciences, Piscataway NJ) and proteins were visualized by e
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20 enhanced chemiluminescence (ECL, Amersham Biosciences).
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22 Northern blot analysis.
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1 Total RNA was prepared using the RNeasy mini kit according to the manufacturer’s
2 instructions (QIAGEN, Valencia CA). Total RNA (5-20 µg as indicated) was electrophoresed on
3 a denaturing 1 % agarose gel and capillary blotted onto a nylon membrane using a Turbo blotter
4 (Schleicher & Schuell, Inc., Keene NH). After UV cross-linking, the membrane was stained for
5 ribosomal RNA (rRNA). For that purpose the membrane was soaked in 5% acetic acid for 15
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6 min at ambient temperature and then incubated in a solution of 0.5 M sodium acetate (pH 5.2) o
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7 and 0.04% methylene blue for 5 min at ambient temperature. The membrane was rinsed in water lo
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8 for 10 min and the stained membrane was photographed. Following staining, the membrane was e
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9 pre-hybridized with 10 ml of QuickHyb Hybridization Solution (Stratagene, La Jolla CA) for 1 h o
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10 at 65oC. The membrane was then hybridized with probes for 5 h at 65oC. The probe for A3F ht
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11 mRNA was a 807 bp EcoRI/HindIII restriction fragment isolated from pcDNA-A3F. Probes /jv
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12 were labeled with [32P]deoxy-TTP and random primer using a Ladderman Labeling Kit (TakaRa, s
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13 Madison WI) and 1 x 107 cpm of the probe was added after premixing with 100 µl sonicated or
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14 salmon sperm DNA (Stratagene, La Jolla CA), heating at 94 ˚C for 5 min, and chilling on ice.
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15 Following hybridization, membranes were washed twice with wash buffer (2 x SSPE, 0.1 % p
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16 SDS) for 15 min at ambient temperature, followed by a 15 min wash in 0.2 x SSPE, 0.1% SDS at ,

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17 60oC. RNA bands were visualized by autoradiography. 1
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19 Virus preparation. e
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20 Virus stocks were prepared by transfection of HeLa cells with appropriate plasmid
21 DNAs. Virus-containing supernatants were harvested 24 to 48 h after transfection. Cellular
22 debris was removed by centrifugation (5 min, 1500 rpm) and clarified supernatants were filtered
23 (0.45 µM) to remove residual cellular contaminants. For immunoblot analyses, filtered virus