A novel role for APOBEC3: Susceptibility to sexual transmission of murine acquired immunodeficiency virus (mAIDS) is aggravated in APOBEC3 deficient mice
© Jones et al.; licensee BioMed Central Ltd. 2012
Received: 14 March 2012
Accepted: 16 May 2012
Published: 12 June 2012
APOBEC3 proteins are host factors that restrict infection by retroviruses like HIV, MMTV, and MLV and are variably expressed in hematopoietic and non-hematopoietic cells, such as macrophages, lymphocytes, dendritic, and epithelia cells. Previously, we showed that APOBEC3 expressed in mammary epithelia cells function to limit milk-borne transmission of the beta-retrovirus, mouse mammary tumor virus. In this present study, we used APOBEC3 knockout mice and their wild type counterpart to query the role of APOBEC3 in sexual transmission of LP-BM5 MLV – the etiological agent of murine AIDs (mAIDs).
We show that mouse APOBEC3 is expressed in murine genital tract tissues and gametes and that genital tract tissue of APOBEC3-deficient mice are more susceptible to infection by LP-BM5 virus. APOBEC3 expressed in genital tract tissues most likely plays a role in decreasing virus transmission via the sexual route, since mice deficient in APOBEC3 gene have higher genitalia and seminal plasma virus load and sexually transmit the virus more efficiently to their partners compared to APOBEC3+ mice. Moreover, we show that female mice sexually infected with LP-BM5 virus transmit the virus to their off-spring in APOBEC3-dependent manner.
Our data indicate that genital tissue intrinsic APOBEC3 restricts genital tract infection and limits sexual transmission of LP-BM5 virus.
KeywordsAPOBEC3 murine AIDS LP-BM5 virus MLV ♀: female ♂: male
Viruses that use the genital mucosa as a portal of entry into the host include HPV, HSV, hepatitis B virus, and retroviruses like SIV, MLV, and HIV. Worldwide, HIV infection is pandemic in many countries and is primarily transmitted from infected persons to their sexual partners through contact with infected semen and vaginal secretions [1–4]. The control of sexual transmission is, therefore, pivotal to curbing the HIV pandemic, especially since the genital tract may also serve as a sanctuary in which HIV undergoes selective pressures [5–8] that may result to the emergence of drug-resistant HIV variants that may be sexually transmitted [9–11]. Indeed, it is known that patients with highly active anti-retroviral therapy (HAART) - suppressed plasma viral load continue to shed viral RNA through the genital tract [12, 13]. It is therefore critical to determine whether host factors can control virus replication and evolution within the male genital tract. Since we could not use human subjects for these experiments, a mouse model offers a practical alternative. Hence, we utilize the murine acquired immunodeficiency syndrome (mAIDs) model.
mAIDs is a mouse retroviral disease caused by the viral complex LP-BM5 . LP-BM5 is a mixture of murine leukemia viruses comprised of the replication competent helper virus BM5e, the mink cell cytopathic focus-inducing virus, and the replication-defective virus BM5def, responsible for the immunodeficiency [15, 16]. Infection of susceptible mice with LP-BM5 causes severe immunodeficiency syndrome similar to human AIDS [14, 17]. Some of the similar characteristics between mAIDS and hAIDS include hypergammaglobulinemia, lymphadenopathy, severely depressed T- and B-cell responses to mitogens, increased susceptibility to infection, disease progression and the development of B-cell lymphomas and splenomegaly [18–22]. Despite the similarities between mAIDS and hAIDS, differences between the two infectious diseases exist. Unlike human AIDS, mice suffering from mAIDS develop mediastinal lymph node enlargement and may die of respiratory failure . Propagation of mAIDS in mice requires the matrix (MA) and p12 of the Gag gene of BM5def . Due to the similarity in virus transmission and disease outcome between LP-BM5- and HIV infections, mAIDs is a good model for examining sexual transmission of HIV and have been used for the initial evaluation of new drugs [25–29].
To be transmitted sexually, HIV and LP-BM5 will have to overcome host resistance in genital mucosal surfaces which are composed of different types and layers of epithelial cells and mucosa-associated lymphoid tissues (MALTs). The male and female genital mucosal layers are filled with cells of the immune system, including dendritic cells (DCs), macrophages and lymphocytes [30–36]. The immune cells in the genital mucosal surfaces express pattern recognition receptors (PRRs) such as Toll-like receptors, RIG-I-like receptors, and NOD-like receptors [37–40] that function to recognize different pathogen associated molecular patterns (PAMPs) and stress signals leading to activation of the host innate and adaptive immune responses. Aside from innate and adaptive immune responses elicited by cells in the genital mucosal surfaces, the expression of anti-viral genes such as APOBEC3 (A3) in male reproductive organs has been reported [41, 42].
A3 proteins are virus restriction factors that encode DNA-editing enzymes. The human genome encodes 7 A3 genes (A3 –A, -B, -C, -D/E, -F, -G, -H), and the mouse genome encodes only 1 A3 gene . A3 proteins are variably expressed in hematopoietic and non-hematopoietic cells, and in different tissues [42, 44–47]. The most studied human A3 genes (A3G and A3F) and mouse A3 (mA3) are reported to confer intrinsic immunity to HIV-1 and other viruses including MMTV and MLV [45–52]. In the absence of HIV-1 viral infectivity factor (Vif), A3F and A3G are packaged into progeny virions via interactions with the nucleocapsid (NC) protein and viral RNA. A3 proteins then inhibit infection in target cells by deaminating deoxycytidine residues on the DNA minus strand following reverse transcription and inducing mutations in newly synthesized HIV-1 DNA. In addition, A3F and A3G degrade reverse transcribed viral DNA prior to integration and induce mutation of viral genes in the integrated provirus [53–56]. However, in the presence of Vif, A3F and 3G are marked for proteasomal degradation [57–59], thus preventing their packaging into progeny virions; resulting in productive infection of target cells. Furthermore, there is growing evidence that A3 proteins can restrict HIV and other viral infection in a deamination independent manner [45, 46, 60, 61]. We previously showed that endogenous mA3 restricts MMTV infection in vivo in a deamination independent mechanism . In addition to limiting virus replication, we recently demonstrated that the rate of milk-borne transmission of MMTV was significantly increased in mice harboring targeted mutation in the mA3 gene , demonstrating that A3 proteins play a role in containing milk-borne virus transmission.
In the present study, we define the role of A3 in sexual transmission of retroviruses using the mAIDs model and mice with targeted mutation in the mA3 gene. Our data indicate that mice with targeted mutation of APOBEC3 gene have a higher rate of sexual transmission compared to the wild type control, suggesting a role for APOBEC3 in limiting sexual transmission.
mA3 is expressed in genital tract tissues and gametes
mA3-deficient mice have increased susceptibility to acute LP-BM5 infection
mA3 deficiency exacerbates mAIDs-associated lympho-proliferation and splenomegaly
Mice lacking mA3 gene have higher genital infection and seminal plasma virus load
Loss of APOBEC3 gene facilitates efficient sexual transmission of LP-BM5
Female mice sexually infected with LP-BM5 transmit the virus to their off-spring in APOBEC3-dependent manner
mA3 genotype permutation for the generation of off-springs from parents of different genetic backgrounds
Off-spring mA3 genotype
♂ 159 -/-
♀ 159C +/-
♂ 160 +/-
♀ 160C -/-
♀ 160D WT
♂ 161 WT
♀ 161D +/-
Genital organ mA3 determines susceptibility to sexual transmission
HIV is mostly propagated worldwide through sexual transmission. While a number of host factors have been identified to enhance sexual transmission of HIV , factors that limit sexual transmission of HIV have not been described. However, APOBEC3 as an anti-HIV factor that restricts infection of hematopoietic and non-hematopoietic cells [47, 50] could potentially play a role in sexual transmission. Recently, in vivo studies in mice show that APOBEC3 restricts MMTV infection of the lymphoid compartment, resulting in lower levels of virus infection in mammary tissue, accompanied by a decreased rate of milk-borne transmission of MMTV .
Here, we demonstrate a possible role for APOBEC3 in restricting a sexually transmitted virus infection. We show that mice with targeted deletion of the Apobec3 gene have higher rates of LP-BM5 infection in lymphoid and genital tissues, and that this results in enhanced rates of sexual transmission of LP-BM5 virus. It is known that LP-BM5 infects genital tissues  and causes severe immunodeficiency and splenomegaly [18–22]. APOBEC3 is made in genital organs; interestingly, the absence of APOBEC3 in these organs, as in APOBEC3 knockout mice, altered infection of the genital organs. Higher level of infection in APOBEC3 knockout mice provides evidence that APOBEC3 mediates the susceptibility of the genitalia and gametes to virus infection. Additionally, the significantly higher vRNA in genital organs of APOBEC knockout mice (Figure 4I), as well as high levels of extracellular vRNA and protein in seminal plasma (Figure 4E and 4G) from APOBEC3 knock mice indicates that LP-BM5 is actively replicating more efficiently in the absence of APOBEC3. Our finding is in line with published reports [75, 76] which showed the occurrence of active virus replication in semen of HIV-1 infected men. The alteration of virus infection in genital compartment in APOBEC3 knockout mice positively correlates with high level of viral RNA in seminal plasma, as well as increased infectivity of seminal plasma LP-BM5 virus compared to wild type mice. The demonstration that extracellular virions from seminal plasma obtained from APOBEC3 knockout mice are more infectious than that from control mice further confirms that loss of mA3 results in higher virus load (Figure 4H). These results suggest that genital organ-intrinsic APOBEC3 expression serves to limit sexual transmission of LP-BM5, and perhaps other retroviruses. Indeed, we show that seminal fluid of APOBEC3 knock mice harbors more LP-BM5 virus compared to the wild type as visualized by Western blot (Figure 4G), but we were unable to detect APOBEC3 due to perhaps the lack of good anti-mouse APOBEC3 antibodies or limited viral protein in samples from APOBEC3 sufficient mice. The detection of LP-BM5 proviral DNA in spermatozoa and testes support previous observations made with HIV-1 and herpesvirus type 8 [77–80].
Using genetic studies, we found that APOBEC3 mediates rate of sexual transmission of LP-BM5. The different permutations of genetic backgrounds used clearly show that the APOBEC3 gene is critical in determining the outcome of sexual transmission of LP-BM5 from one partner to the other, and from a sexually infected mother to her off-spring through nursing. Although loss of APOBEC3 enhanced rate of sexual transmission, we also observed that APOBEC3 deficient female mice were more highly infected compared to the heterozygote and wild type controls in that order. These genetic experiments show that APOBEC3 also plays a crucial role in mediating virus acquisition from an infected male and that the female genetic background contributes in defining the outcome of infection.
We also used adoptive transfer and in vivo genetic experiments to demonstrate a clear relationship between genital-intrinsic APOBEC3 and sexual transmission. Genital-intrinsic APOBEC3 mediates control of sexual transmission since higher levels of splenocyte infection observed in male mice that received APOBEC3 knockout cells did not translate to higher rate of female partner infection. This concept was further confirmed by similar level of infection observed in the off-springs of these mice irrespective of the male parent splenocyte APOBEC3 genotype.
While murine retroviruses (beta and gamma retroviruses) are sensitive to A3 antiviral activity, they have also successfully replicated in mice. It is not clear how these infectious viruses have evaded host restriction to achieve efficient replication, since they do not encode viral accessory proteins, such as the HIV-1 Vif protein that antagonizes some human A3 proteins. However, reports of alternative mechanisms used by murine retroviruses to evade restriction by A3 proteins exist. Indeed, MLV virions have been shown to exclude mA3  as well as inactivate mA3 by viral protease , and xenotropic murine leukemia-virus related virus (XMRV) down-regulates hA3G in prostate cancer cells .
Sexual transmission of HIV-1 continues to be a problem worldwide, especially in developing countries, where access to condoms is restricted by cultural and socio-economic situations. Previous studies have indicated that the use of HAART in the control of HIV transmission has mixed results since some HAART treated patients with suppressed plasma viral load continue to shed viral RNA in semen [12, 13].
As in immune cells and mammary epithelia cells, APOBEC3 is made in genital organs and gametes. Based on our data, we suggest that APOBEC3 expression in genital organs and gametes may have been retained as a means of inhibiting sexual transmission of viruses. In addition, APOBEC3 in genital organs could have important therapeutic implications for sexual transmission of viruses. For example, therapeutics could be developed that increase APOBEC3 level in genital organs, resulting in attenuated virions.
Experiments involving the use of mice were performed in accordance to NIH guidelines, the Animal Welfare Act, and US federal law. Experiments were approved by the University of Iowa Animal Care and Use Committee (IACUC).
MEF and primary testicular cells were obtained from mA3-/-, mA3+/-, and WT mice on C57BL/6 background. SC1-G6 cells chronically infected with LP-BM5 (containing ecotropic, mink cell focus-forming and defective viruses) were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr. Herbert Morse and Dr. Janet Hartley [15, 19, 23].
The mA3-knockout (mA3-/-) and matched controls mA3+/-, (mA3+/+, WT) mice have been previously described . All mice were housed according to the policies of the Institutional Animal Care and Use Committee of the University of Iowa.
Five age-matched (5 weeks old) male mice of each genotype were lethally irradiated with gamma rays. All radiation experiments were performed at the Radiation and Free Radical Research Core Facility of the University of Iowa. Mice were irradiated with a dose of 9 Gy (dose rate, 0.345 Gy/min) using a 137Cs source (JL Shepherd, San Fernando, CA) as previously described . Four hours after irradiation, mice were reconstituted with bone marrow stem cells from either mA3-/- or WT mice. Four weeks after reconstitution, the mice were inoculated with LP-BM5 virus. Prior to infection, peripheral blood lymphocytes from each mouse were subjected to FACs analysis with conjugated anti-mouse CD8, CD4, CD11c and B220, to test for uniform reconstitution.
Cell-free LP-BM5 viral mixture was prepared from chronically infected SC-l cells as previously described . Culture supernatants were collected and centrifuged at 1500 rpm for 10 minutes to clarify cellular debris. Clarified culture supernatants were filtered through a 0.45 μM filter, layered on to 20% sucrose cushion and centrifuged at 7000 rpm, 4 °C overnight. Virus pellet was resuspended in PBS at 10:1 ratio and aliquots of 100 μl stored at -80 °C until required for use.
Cell and mouse infection
Infection of cells with cell culture or seminal plasma virus was done in triplicates in 96-well plate format. Briefly, primary testicular cells or splenocytes were plated at ~100,000 cells per well. Twenty four hours later, cells were incubated with 8 μg/ml of polybrene for 2 hours. Polybrene-containing medium was removed and replaced with 100 μl of virus-containing medium or PBS for control. Cells were spinoculated at 1200 rpm for 2 hours at room temperature. Following spinoculation, cells were incubated at 37 °C for 24 hours. DNA was extracted from cells for examination of viral load. Mice infections were done either subcutaneously (SubQ) on the hind footpad or intraperitoneally (IP). For footpad inoculation, 30 μl of virus or PBS was injected into the footpad tissue and IP inoculation was performed with 200 μl of virus or PBS. At the appropriate time depending on experiment, mice were sacrificed and relevant tissues obtained for downstream analysis.
Genotyping mA3 -/- mice
Mice genotypes were determined using two parallel PCRs as previously described . Briefly, DNA was extracted and 50 ng of genomic DNA and 25 pmol of primers were used in a total volume of 25 μl PCR reaction. PCR amplicons were separated on a 2% agarose gel.
Nucleic acid isolation and real-time quantitative PCR
Milk and seminal plasma viral loads were determined for BM5def as previously described . Briefly, total RNA was isolated from seminal fluid (200 μl) or milk (50 μl) samples using Trizol RNA Reagent (AMRESCO). Isolated RNA was DNAsed and subjected to cDNA synthesis (Applied Biosystems, ABI) as previously described . DNA was also isolated from different tissues using QIAGEN-QIAamp DNA Mini Kit following manufacturer’s recommendation. Nucleic acid concentration and purity were measured spectrophotometrically at 260/280 nm. DNA and cDNA were amplified with primers and probe specific to BM5def  and GAPDH using ABI 7500 FAST thermal cycler (ABI).
Approximately, 1 × 106 splenocytes were stained in PBS + 1% bovine serum albumin (Sigma-Aldrich) for 30 minutes on ice with APC-, FITC-, and PE-conjugated antibodies and the resulting immunofluorescence was analyzed by use of a FACSCalibur flow cytometer (BD) to detect the expression of murine CD69, B220, and CD4 (BD or eBioscience). Cellular frequency and fluorescence intensity were determined by Flowjo analysis software (TreeStar). APC-, FITC-, and PE-conjugated Ig isotypes of irrelevant specificity were used as control for each monoclonal antibody.
Western blots of virus preparations and cell extracts were probed with anti-p30 (MLV capsid) and anti-GAPDH. The species-appropriate IRDye secondary antibodies were used, followed by detection with the Odyssey Infrared Imaging System (LI-COR Biosciences).
Virus purification from seminal plasma
The seminal samples obtained from epididymis were processed immediately and pooled within genotype to provide sufficient seminal plasma volume and seminal cells. Pooled epididymal seminal samples were centrifuged for 15 minutes at 400 g. The pellet was further purified to obtain pure sperm cells as discussed below. The supernatant was collected and filtered through a 0.45 μM filter. Virions were purified from the supernatants by ultracentrifugation at 40,000 rpm for 30 minutes. Viral pellet was resuspended in PBS and stored in 50 μl aliquots at -80 °C until required for use.
Pooled epididymal seminal samples were subjected to standard swim-up technique to purify sperm cells. Briefly, semen samples were centrifuged for 15 min at 400 g. The pellets were resuspended in pre-warmed 2.5 ml Ham's F-10 culture medium (Sigma) supplemented with 10% human serum albumin. Cells were layered over equivalent volume of 80% Percoll and 40% Percoll and centrifuged for 15 minutes at 400 g as previously described . The supernatant was discarded and the pellet was resuspended in Ham's F-10 culture medium and thereafter centrifuged for 15 minutes at 400 g. The supernatant was removed and the pellet was overlaid with 2.5 ml Ham's F-10 medium and incubated for 60 min in 5% CO2 at 37 °C to allow spermatozoa to swim up from the pellet. A sterile Pasteur pipette was used to remove the supernatant containing actively motile sperms. A drop of the sample was examined under light microscope and images acquired with Nikon Eclipse Ti (Nikon Instruments). Purified cells were stored at -80 °C until required.
The paired two-tailed Student's t test was used for statistical analysis and a p value of 0.05 was considered significant. Error bars represent standard deviations.
human immunodeficiency virus
murine leukemia viruses
murine acquired immunodeficiency syndrome.
This research was supported by University of Iowa Startup funds to CMO. PHJ was supported by Immunology T32 training grant to the University of Iowa. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: SC-1/MLV LP-BM5 from Dr. Herbert Morse and Dr. Janet Hartley. Radiation experiments were made possible by the Radiation and Free Radical Research Core Facility of the University of Iowa and Holden Comprehensive Cancer Center Support Grant P30 CA086862. We would like to thank Bryson Okeoma for his analytical critique of this manuscript.
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