Tree Shrew Cells Transduced with Human CD4 and CCR5 Support Early Steps of HIV-1 Replication, but Viral Infectivity Is Restricted by APOBEC3

ABSTRACT The host range of human immunodeficiency virus type 1 (HIV-1) is narrow. Therefore, using ordinary animal models to study HIV-1 replication, pathogenesis, and therapy is impractical. The lack of applicable animal models for HIV-1 research spurred our investigation on whether tree shrews (Tupaia belangeri chinensis), which are susceptible to many types of human viruses, can act as an animal model for HIV-1. Here, we report that tree shrew primary cells are refractory to wild-type HIV-1 but support the early replication steps of HIV-1 pseudotyped with the vesicular stomatitis virus glycoprotein envelope (VSV-G), which can bypass entry receptors. The exogenous expression of human CD4 renders the tree shrew cell line infectible to X4-tropic HIV-1IIIB, suggesting that tree shrew CXCR4 is a functional HIV-1 coreceptor. However, tree shrew cells did not produce infectious HIV-1 progeny virions, even with the human CD4 receptor. Subsequently, we identified tree shrew (ts) apolipoprotein B editing catalytic polypeptide 3 (tsAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity, with virus infectivity reduced 10- to 1,000-fold. Unlike human APOBEC3G, the tsA3Z2c-Z1b protein was not degraded by the HIV-1 viral infectivity factor (Vif) but markedly restricted HIV-1 replication through mutagenicity and reverse transcription inhibition. The pooled knockout of tsA3Z2c-Z1b partially restored the infectivity of the HIV-1 progeny. This work suggests that tsAPOBEC3 proteins serve as an additional barrier to the development of HIV-1 tree shrew models, even when virus entry is overcome by exogenous expression of human CD4. IMPORTANCE The development of animal models is critical for studying human diseases and their pathogenesis and for evaluating drug and vaccine efficacy. For improved AIDS research, the ideal animal model of HIV-1 infection should be a small laboratory mammal that closely mimics virus replication in humans. Tree shrews exhibit considerable potential as animal models for the study of human diseases and therapeutic responses. Here, we report that human CD4-expressing tree shrew cells support the early steps of HIV-1 replication and that tree shrew CXCR4 is a functional coreceptor of HIV-1. However, tree shrew cells harbor additional restrictions that lead to the production of HIV-1 virions with low infectivity. Thus, the tsAPOBEC3 proteins are partial barriers to developing tree shrews as an HIV-1 model. Our results provide insight into the genetic basis of HIV inhibition in tree shrews and build a foundation for the establishment of gene-edited tree shrew HIV-1-infected models.

melanoma differentiation factor 5 (MDA5), which is a PRR and shares similar signaling features and structural homology as RIG-I, can functionally substitute for RIG-I in tree shrews to sense RNA viruses and induce type-1 interferon (IFN-1) response (37,40), delayed IFN-1 expression renders tree shrews more susceptible to viral infection. Our previous work showed that tree shrew TRIM5 proteins, including TRIMCyp, are unable to restrict HIV-1, unlike rhesus monkey TRIM5, which impacts macaque HIV-1 models by delivering potent anti-HIV-1 activity (39). In addition, we identified five tree shrew (ts) APOBEC3 family proteins that induce G-to-A hypermutation in HIV-1 genome DNA (35). However, whether HIV-1 Vif can degrade tsAPOBEC3 proteins, as it does to human APOBEC3, remains unclear. Investigating this could help determine if tree shrews can serve as a potential HIV-1 animal model.
Here, we showed that tree shrew primary cells are refractory to replication-competent HIV-1 but support vesicular stomatitis virus glycoprotein envelope (VSV-G)-HIV-1-green fluorescent protein (GFP) infection, which bypasses the cell membrane receptors. The exogenous expression of human CD4 or CD4/CCR5 in tree shrew lung fibroblasts (TSLFs) also supports HIV-1 entry and efficient infection. However, the virions produced from the human-receptor-expressing TSLFs exhibit low infectivity. Furthermore, the tsAPOBEC3 proteins are potent inhibitors of HIV-1 infection via the insertion of hypermutations and restriction of reverse transcription. Moreover, tsA3Z2c-Z1b is not degraded by HIV-1 Vif. Hence, multiple restrictions exist against HIV-1 replication in tree shrew cells. Our study provides information regarding intrinsic immune restriction factors in tree shrews and essential guidance for developing a tree shrew HIV-1 infection model.

RESULTS
Primary tree shrew cells can be infected by HIV-1 (VSV-G). To evaluate tree shrew potential as an animal model of HIV-1, we first determined if tree shrew cells could support the early phases of HIV-1 replication. Primary tree shrew cells (e.g., lung, spleen, heart, and kidney cells from adult or newborn tree shrews) were subjected to infection with single-cycle VSV-G-pseudotyped HIV-1-GFP virus, which bypasses the HIV-1 cellular receptor. The Crandell-Rees feline kidney (CRFK) cell line was used as a positive control, and owl monkey (Aotus trivirgatus) kidney (OMK) epithelial cells, which express nonpermissive TRIMCyp, were used as a negative control (41). At 3 days postinfection (dpi), the percentage of GFP-positive cells was examined to indicate the permissivity of infection. As shown in Fig. 1A, all primary cells from adult and newborn tree shrews had GFP percentages similar to that in CRFK cells, whereas almost no OMK cells were GFP positive. In addition, the primary tree shrew cells were permissive for SIV-GFP expression (Fig. 1B). The GFP signal percentage was high in splenic and lung cells but low in kidney cells.
To further confirm whether primary tree shrew cells were susceptible to retrovirus transduction, VSV-G HIV-1 NL4-3 , a full-length replication-competent HIV-1, was used to infect tree shrew cells. Again, primary cells from the heart, lung, and kidney were used, and HeLa cells with or without HIV-1 protease inhibitor indinavir (IDV) 100 nM were used as controls to confirm the infection system. Results showed that HIV-1 p24 proteins were as efficiently expressed in the culture of primary tree shrew cells as that of HeLa cells (Fig. 1C). Furthermore, tree shrew peripheral blood mononuclear cells (PBMCs) produced HIV-1 p24 equivalent to that found in human PBMCs (Fig. 1D), suggesting that the long terminal repeat (LTR)-driven transcription of HIV-1 was processed efficiently in the tree shrew cells. The expression levels of virus protein p24 in cells and virions produced from cells infected with the highest virus volume were detected by Western blotting. Results showed that IDV limited HIV-1 expression in the tree shrew (ts) PBMCs (Fig. 1E). Whole DNA was then extracted from these cells for the detection of reverse transcription (ssDNA) using real-time quantitative PCR (RT-qPCR). As shown in Fig. 1F, the tsPBMCs displayed comparable ssDNA signals as those of the human PBMCs. This ensured that the VSV-G HIV-1 NL4-3 virus entered the tree shrew cells and successfully commenced reverse transcription.
Whether primary tree shrew cells or PBMCs can be infected by replication-competent HIV-1 IIIB or NL4-3 was examined. Human cell lines or PBMCs were used as the positive control, and CRFK or rhesus monkey PBMCs were used as the negative control. Results showed that p24 expression in the culture supernatant of tree shrew cells was almost undetectable, similar to that in the CRFK and rhesus monkey controls (Fig. 1G to J). The expression levels of virus protein p24 in the infected cells and virions produced from cells with the highest multiplicity of infection (MOI) were detected by Western blotting.
Collectively, these results suggest that tree shrew cells efficiently support the postentry steps of HIV-1 infection but lack a functional receptor.
HIV-1 infection and integration into tree shrew lung fibroblasts expressing exogenous human CD4 and CCR5. The CD4 receptor protein and CCR5 and CXCR4 coreceptors are critical for HIV-1 infection and act as barriers to restrict HIV-1 adhesion and entry into target cells. Research has shown that macaque CD4 supports HIV-1 entry but mouse CD4 does not (8,42). To examine whether HIV-1 receptors are barriers to cross-species transmission in tree shrews, we aligned the tree shrew CD4, CCR5, and FIG 1 Infection of primary tree shrew cells with VSV-G HIV-1 but not with wild-type HIV-1. (A and B) Primary tree shrew cells (e.g., lung, spleen, heart, and kidney from adult [marked as A] or newborn [marked as N] tree shrews) were subjected to infection with various doses of single-cycle VSV-G-pseudotyped HIV-1-GFP virus (A) and SIVmac-GFP virus (B). (C and D) At 3 dpi, percentages of GFP-positive cells were determined by flow cytometry. Tree shrew primary cells (C) and tree shrew, human, and rhesus monkey PBMCs (D) were infected with VSV-G HIV-1 NL4-3 . Expression levels of p24 in supernatants were then analyzed at 3 dpi by ELISA. (E) tsPBMCs were infected by VSV-G HIV-1 NL4-3 with IDV. At 30 hpi, expression of p24 in supernatants was analyzed. (F) At 4 hpi with VSV-G HIV-1 NL4-3 , ssDNA signals were detected by RT-qPCR in PBMCs. (G to J) HIV-1 IIIB was used to infect primary tissue cells (G) or PBMCs (H); HIV-1 NL4-3 was used to infect primary tissue cells (I) or PBMCs (J), with p24 expression levels in the supernatant analyzed at 3 dpi. Virus proteins of infected cells and virions (infected with highest virus volume or MOI) were detected by Western blotting. HeLa (IDV) and human (IDV) are HIV-1 protease inhibitor indinavir controls, which were added to confirm the infection system. CXCR4 amino acid sequences with the orthologous sequences in humans and macaques. As shown in Fig. 2, the homologies between tree shrew and human CD4, CCR5, and CXCR4 were 61.0%, 78.1%, and 97.7%, respectively (aligned by ClustalW; https:// www.genome.jp/tools-bin/clustalw), suggesting that CD4 and CCR5 may be limiting factors for HIV-1 infection in tree shrew cells.
To reconstruct tree shrew cell lines that express functional receptors for HIV-1, we first obtained TSLFs. We confirmed whether TSLFs could support the early phases of HIV-1 replication by determining the percentage of GFP-positive cells after single-cycle VSV-Gpseudotyped HIV-1-GFP virus infection (Fig. 3A). We then stably transduced TSLFs with retroviral vectors expressing human CD4 and CCR5. Three cell lines were generated, including single receptor-bearing TSLF-CD4 and TSLF-CCR5 and dual receptor-bearing TSLF-CD4-CCR5 cells. Flow cytometry confirmed the cell surface expression of human CD4 and CCR5 in the TSLF, TSLF-CD4, TSLF-CCR5, and TSLF-CD4-CCR5 cells (Fig. 3B).
tsCXCR4 is a functional coreceptor for HIV-1 entry. We next examined whether tsCXCR4 is functional for HIV-1 entry. First, we performed flow cytometry with an anti-human CXCR4 monoclonal antibody to examine CXCR4 expression in TSLFs. Results showed that four types of TSLFs expressed the CXCR4 chemokine receptor at detectable levels by the anti-human CXCR4 antibody, though the expression levels were lower than that in HOS cells (Fig. 4A). Second, plerixafor (AMD3100), a CXCR4 inhibitor, and maraviroc (MVC), a CCR5 inhibitor, were used as controls (3mM). Results showed that AMD3100 reversed the X4tropic HIV-1 IIIB infection in permissively engineered tree shrew cells, with the p24 level in the supernatant almost 1,000-fold lower than that in the untreated cells or MVC control (Fig. 4B).
In addition, we knocked down tsCXCR4 expression in TSLFs expressing human CD4 or CD4-CCR5 (Fig. 4C). The CXCR4 envelope HIV-1 NL4-3-Luc -pseudotyped virus was used for infection. As shown in Fig. 4D, the luciferase signal was significantly lower in the tsCXCR4 knockdown cells than in the control cells. These findings indicate that tsCXCR4 is a coreceptor for HIV-1 adhesion and entry.
Impaired viral infectivity of HIV-1 progeny from TSLF-CD4-CCR5 cells. We next evaluated the infectivity of the progeny virus produced from the receptor-engineered tree shrew cells. A high multiplicity of infection (MOI) (1.0) of replication-competent HIV-1 Bal or HIV-1 IIIB was used to infect target tree shrew cells. At 48 h postinfection (hpi), the cell culture supernatant was normalized to 0.3 ng of p24 for the infection of TZM-bl reporter cells. We showed that luciferase activity in the TZM-bl cells infected with the supernatant from the receptor-engineered tree shrew cells was close to that of the negative control infected with the TSLF supernatant but much lower than that of TZMbl cells infected with the HOS-CD4-CCR5 supernatant. Thus, the progeny viruses from the tree shrew TSLF-CD4 and TSLF-CD4-CCR5 cells were likely deficient for reinfection (Fig. 5A). To exclude the possibility that cell species may affect progeny virus infectivity, we infected other cells with 0.3 ng of p24 from the culture supernatant from the first-round infection. As shown in Fig. 5B to D, only the progeny virus from HOS-CD4-CCR5 cells could reinfect HOS-CD4-CCR5, TSLF-CD4-CCR5, and TZM-bl cells, showing detectable p24 levels in the supernatant and integrated proviral DNA in the cells, whereas viruses produced from the TSLF or TSLF-CD4-CCR5 cells were noninfectious, even when a larger input load of progeny virus was used to reinfect the cells (Fig. 5E). In addition, in the cell-to-cell infection assay, luciferase activity was undetectable in TSLF-CD4-CCR5 cells cocultured with TZM-bl but not in HOS-CD4-CCR5 cells cocultured with TZM-bl (Fig. 5F).
With the observation that tree shrew cells were permissive to the VSV-G HIV-1-GFP reporter virus but produced low-infectivity virions, we next investigated the underlying molecular basis. We first characterized the progeny virus genome from the infected tree shrew cells. We extracted genomic RNA from the culture supernatant and then amplified the full-length genome. As detected by agarose gel electrophoresis, there were no obvious differences between the progeny virus genome from the HOS-CD4-CCR5 cells and the TSLF-CD4-CCR5 cells (Fig. 5G). Additionally, there was no severe mutation in the progeny virus genome sequence from the TSLF-CD4-CCR5 cells (data not shown). The progeny virus from the TSLF-CD4-CCR5 cells was as morphologically normal as that from the chronically infected H9 cells and could enter target cells successfully (Fig. 5H). However, we found that the viral products of early and late reverse transcription, as well as the products of viral nucleus transport, were impaired in C8166 cells infected by the progeny virus from TSLF-CD4-CCR5 compared to C8166 cells infected by the progeny virus from HOS-CD4-CCR5 (Fig. 5I). Furthermore, the subsequent integration process was not detectable (Fig. 5J). Overall, these findings showed that the infectivity of the progeny viruses produced from the receptor-engineered tree shrew cells was greatly impaired.
tsAPOBEC3 proteins inhibit infectivity of HIV-1 progeny. The above results suggest that tree shrew cells may express certain restriction factors that affect the production of infectious HIV-1 progeny virus. It has been reported that human APOBEC3G (huA3G) can be packaged into viral particles and induce G-to-A hypermutations in the viral genome during reverse transcription in the target cells (43,44). To counteract huA3G, HIV-1 produces a protein called Vif to degrade huA3G and therefore exclude huA3G packaging in the virions (45,46). We previously identified five tsAPOBEC3 member proteins that could induce G-to-A and C-to-T hypermutations in the HIV-1 genome (35). Based on this observation, we wondered if tsAPOBEC3 proteins play a part in the restriction of HIV-1. In the TSLF-CD4-CCR5 HIV-1 infection system, we found that tsAPOBEC3 proteins were expressed in TSLF cells (Fig. 6A).
As the suppression of reporter proteins is correlated with a significant increase in G-to-A hypermutations in viral reverse transcription products or with restricted elongation of reverse transcripts in target cells (52), we detected whether the viral products were affected by tsAPOBEC3 proteins. Progeny viruses produced from 293T cells cotransfected with tsAPOBEC3 and pNL4-3-Dvif plasmids were first used to infect C8166 cells. Whole DNA of the C8166 cells was then extracted for the detection of ssDNA, completion of reverse transcription (lateRT), and transportation to the nucleus (2LTR) by RT-qPCR. Results showed that tsA3Z2c-Z1b strongly inhibited reverse transcription, late reverse transcript production, and nuclear transport (Fig. 6H).
Depletion of tsA3Z2c-Z1b increases infectivity of progeny virus from tree shrew cells. The above findings showed that tsA3Z2c-Z1b has strong anti-HIV activity when overexpressed. To explore the effects of tsA3Z2c-Z1b on the infectivity of progeny virus in vitro, we generated a pooled tsA3Z2c-Z1b-knockout (KO) TSLF-CD4 cell line using CRISPR/Cas9. The tsA3Z2c-Z1b genome was sequenced in the tsA3Z2c-Z1b-KO mixed TSLF-CD4 cells. The tsA3Z2c-Z1b gene was edited to form a frameshift mutation using CRISPR/Cas9 (Fig. 7A). As shown in Fig. 7B, when the predicted endogenous expression of tsA3Z2c-Z1b was efficiently reduced as indicated by Western blotting, p24 production was higher than that in the knockout control from the first round of HIV-1 IIIB infection. The progeny virus was then used to reinfect C8166 and TZM-bl cells with normalized 1 ng or 10 ng of p24, respectively. The progeny virus from the tsA3Z2c-Z1b-knockout TSLF-CD4 cells effectively reinfected the C8166 and TZM-bl cells, whereas the progeny virus from the TSLF-CD4 cells lacked infectivity ( Fig. 7C and  D). Thus, the depletion of endogenous tsA3Z2c-Z1b helped to restore the infectivity of the HIV-1 progeny virus, demonstrating its critical role in HIV-1 restriction.
In conclusion, the tsAPOBEC3 proteins, especially tsA3Z2c-Z1b, play an important role in suppressing the infectivity of HIV-1 virions and significantly contribute to the blocking of HIV-1 propagation in TSLF-CD4-CCR5 cells.

DISCUSSION
In this study, we characterized the ability of tree shrew cells to support HIV-1 replication to determine whether tree shrews can be developed into animal models for HIV-1 studies. We found that the exogenous expression of human CD4 and CCR5 molecules in tree shrew cells was sufficient for HIV-1 entry and replication and that tsCXCR4 serves as a functional coreceptor. However, additional blocks to HIV-1 infection were identified in the tree shrew cells. In particular, tree shrew cytidine deaminases of the APOBEC3 family were found to exert a strong inhibitory effect on HIV-1 replication.
Since the discovery of HIV in 1983 as the pathogen of AIDS, our understanding of the virus has greatly improved. However, the development of effective animal models is essential for continued study of the pathogenesis of AIDS and to evaluate the efficacy of drugs and vaccines. The ideal animal model of HIV infection should be a small laboratory mammal that closely mimics virus replication in humans. However, suitable small animal models of HIV-1 do not yet exist, though a variety of feline and humanized rodent models of HIV-1 infection have been developed (1,8,9,(53)(54)(55). The failure of establishing HIV-1 models is primarily related to functional receptors or coreceptors and postinfection host restriction factors. A lack of functional receptors is the first impediment to HIV-1 infection in murine, feline, and ferret cells (1, 8, 56). Restriction factors, on the other hand, constitute the main impediment of HIV-1 replication in simian cells. For example, cytoplasmic-body protein TRIM5a restricts HIV-1 replication at the postentry step in simian cells (57). In contrast, the TRIMCyp fusion protein expressed in pig-tailed macaques exhibits no restriction to HIV-1 activity, identifying a potential molecular mechanism that may explain why pig-tailed macaques are prone to HIV-1 infection (58). Murine and feline cells exhibit additional restrictive factors for HIV-1 replication other than setting receptor limitations: murine cells lack LTR-driven transcription functions (8,55), and feline APOBEC3 proteins restrict HIV-1 replication by inducing G-to-A hypermutation in the viral genome (1).
We previously showed that tree shrews express APOBEC3 proteins that induce Gto-A and C-to-T hypermutations in the HIV-1 genome (35). In the tree shrew hepatitis B virus (HBV) model, tsAPOBEC3 may limit the establishment of HBV-persistent infection due to its strong anti-HBV replication ability (59). Here, we investigated whether tsAPOBEC3 proteins can account for the failure of tree shrew cells engineered with human CD4 and CCR5 receptors to produce infectious particles. The APOBEC3 protein is usually packaged into retroviral particles from the producing cells and is transmitted to the target cells through infection (26). "Nonpermissive" cells exhibit APOBEC3G expression and are resistant to delta Vif HIV-1 replication. To antagonize the restriction of APOBEC3G, HIV-1 encodes the Vif to degrade human APOBEC3 proteins and reduce the incorporation of APOBEC3 into progeny virions (60). HIV-1 is strongly inhibited by simian APOBEC3G and murine APOBEC3 because the charge of amino acid 128 determines its functional interaction with Vif. When this site is positively charged, HIV-1 Vif cannot degrade APOBEC3G (61)(62)(63). In this context, the fact that tsA3Z2c-Z1b was potently restrictive to HIV-1 and not degraded by the Vif may be the cause of the low infectivity of the progeny virus from TSLF-CD4-CCR5, making TSLF-CD4-CCR5 cells nonpermissive and therefore potentially inappropriate as an HIV-1 cell model.
In summary, we observed multiple restrictions against HIV-1 replication in tree shrew cells in this study. We characterized tree shrew membrane receptors and APOBEC3 proteins as limitations that initiate the restrictions. Further detailed analysis of why HIV-1 cannot overcome the cellular barriers of tree shrews or other animals will likely generate knowledge of how to genetically modify human cells against HIV-1 replication and help to better understand how to prevent viral cross-species transmission.

MATERIALS AND METHODS
Collection of tree shrew tissues and isolation of cells. Tree shrews were raised at the Experimental Core Facility of the Kunming Institute of Zoology, Chinese Academy of Sciences. Brain, heart, liver, lung, spleen, colon, and blood samples were isolated from healthy male adult and newborn tree shrews. All experimental procedures were performed according to the guidelines approved by the Ethics Committee of the Kunming Institute of Zoology (approval number SYDW-2015017).
Primary tissue cultures of newborn and adult tree shrews were established by mincing dissected organs, followed by trypsinization for 30 min at 37°C. Cells were washed, filtered through a 40-cm pore tissue culture mesh filter (Falcon), and then grown in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and penicillin and streptomycin.
Infection assays. For single-cycle infection assays, cells were seeded in 24-well plates at a density of 5 Â 10 4 cells/well and incubated overnight at 37°C in 5% CO 2 . The cells were then infected with VSV-Gpseudotyped viruses. At 48 hpi, cells were washed with phosphate-buffered saline (PBS), and then the percentage of GFP-positive cells was examined by flow cytometry.
For the PBMC infection assays, freshly activated PBMCs (5 Â 10 6 cells) were seeded in 24-well plates and infected with HIV-1. At 16 hpi, cells were washed three times with PBS and resuspended in fresh RPMI-10% FBS supplemented with IL-2. Supernatants were collected at 72 hpi. The number of viral particles released into the cell culture supernatant was quantitated by p24 enzyme-linked immunosorbent assay (ELISA; ZeptoMetrix, USA).
For multiple-cycle infection assays, cells were seeded in 12-well plates incubated overnight at 37°C in 5% CO 2 . The cells were then infected with HIV-1 or transfected with HIV-1 expression plasmids, with the culture medium refreshed 6 h later. If an inhibitor was needed, 100 nM IDV or 3 mM AMD3100/MVC was added. IDV was added as a control to detect the infection system. Plerixafor (AMD3100) is a CXCR4 inhibitor, and MVC is a CCR5 inhibitor. After 48 to 72 h, the supernatants and cells were collected to detect p24 by ELISA and virus/cell proteins by Western blot analysis. The supernatants were normalized to the same viral level for reinfecting target cells. After 72 h, the cells or culture supernatants were collected to detect luciferase activity or p24 levels.
For HIV-1 NL4-3-Luc infection assays, cells were seeded in 24-well plates at a density of 5 Â 10 4 cells/well and incubated overnight at 37°C in 5% CO 2 . The cells were then infected with HIV-1 NL4-3-Luc viruses. At 72 hpi, cells were washed with PBS and then collected to detect luciferase activity.
In the flow cell-sorting experiment, mCherry-positive cells were sorted using a Sony SH800 flow cytometer (Japan) driven to gain TSLF-CD4 tsA3Z2c-Z1b knockout cells.
ELISA for quantification of p24 levels. At 48 to 72 hpi, the cultured supernatants were collected and centrifuged at 200 Â g for 5 min at room temperature to remove cell debris. The culture supernatants were analyzed using commercial HIV-1 p24 ELISA kits (Wantai BioPharm, China).
Western blot analysis. Cells were lysed for 30 min on ice and then centrifuged at 13,000 Â g for 10 min at 4°C, with the supernatants then transferred into clean 1.5-ml Eppendorf tubes.
The supernatants were centrifuged at 13,000 Â g for 5 min at room temperature to remove cell fragments 72 h later. After resuspending the precipitation with polyethylene glycol 8000 (PEG 8000) overnight, the supernatants were centrifuged at 13,000 Â g for 30 min at 4°C to collect viral particles. The particles were lysed with lysis buffer for 30 min on ice and then stored at 280°C.
The prepared proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The blots were probed with a primary antibody at 4°C and then a secondary antibody at room temperature for 1 h. Afterward, the blots were illuminated with chemiluminescent detection reagents (Millipore, USA).
The serum of HIV-1-positive patients (obtained from Kunming Third People's Hospital, China) was used to detect glycoprotein 120 (gp120). The mouse anti-p24 polyclonal antibody (produced in-house) was used to detect p24 and p55. The mouse anti-HA monoclonal antibody (catalog no. H3663; Sigma-Aldrich, USA) or anti-Flag monoclonal antibody (catalog no. F1804; Sigma-Aldrich, USA) was used to detect tsAPOBEC3. The mouse hAPOBEC3G monoclonal antibody (ImmunoDiagnostics, USA) was used to detect tsA3Z2c-Z1b.
Quantitative real-time PCR. Total DNA from cells was isolated using a TIANamp genomic DNA kit (Tiangen Biotech, Beijing). For the virus genome, commencement of reverse transcription (ssDNA), completion of reverse transcription (lateRT), and transportation to the nucleus (2LTR) were determined by RT-qPCR, as described previously (65).
Total RNA from cells was isolated and then reverse transcribed into cDNA using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). To quantify tsAPOBEC3 mRNA expression levels, RT-qPCR was performed as per previous study (35).
The following conditions were used for the first round of PCR: 94°C for 8 min; followed by 94°C for 30 s, and 50°C for 45 s; and 72°C for 4 min for 12 cycles and a final extension at 72°C of 10 min. The firstround PCR products were used as a template for the second round of PCR, which was performed with the following conditions: 94°C for 8 min; followed by 94°C for 30 s, 50°C for 45 s, and 72°C for 4 min for 35 cycles; and finally, 10 min at 72°C. The PCR products were detected using agarose gels.
Full-length HIV-1 RNA detection. To detect the progeny virus genome from infected tree shrew cells, we extracted genomic RNA from the culture supernatant of HOS-CD4-CCR5 and TSLF-CD4-CCR5 cells and then amplified the full-length genome. The full-length genome was divided into three sections for detection. The primers for nested PCR and conditions were as before (67).
Transmission electron microscopy. Transmission electron microscopy was used as described previously (68,69). In brief, samples were fixed overnight at 4°C using 2.5% glutaraldehyde in PBS. Afterward, samples were postfixed with 1% OsO 4 at 4°C for 2 h, followed by serial ethanol dehydration and embedding in Epon 812 resin. Serial sections of uniform thicknesses (;60 nm) were made using a Leica EM UC7 ultramicrotome (Germany). Ultrathin sections were then loaded onto 100-mesh Cu grids and double stained with 2% uranyl acetate and lead citrate before observations were made using a JEM 1400Plus transmission electron microscope (Japan) at 120 kV.
Data availability. All sequences reported in this paper were deposited in GenBank as follows: tree shrew CD4, accession no. MN227147; tree shrew CCR5, accession no. MN227148; and tree shrew CXCR4, accession no. MN227149. We declare no conflict of interest.