Alpha-1-antitrypsin interacts with gp41 to block HIV-1 entry into CD4+ T lymphocytes
© The Author(s). 2016
Received: 11 January 2016
Accepted: 15 June 2016
Published: 29 July 2016
Study of a clinic case reveals that alpha-1-antitrypsin (AAT) deficiency is related to CD4+ T cell count decline and AIDS progression, suggesting that AAT might be an endogenous inhibitor of HIV/AIDS. Previous study shows that AAT inhibits HIV-1 replication in infected host cells and the C-terminus fragment of AAT, VIRIP, interferes with HIV-1 infection. However, it is still unclear whether and how intact AAT inhibits HIV-1 infection. It is also unknown what the mechanism of AAT is and which critical step(s) are involved.
In the present study, the C-terminus of AAT (C) was synthesized. C terminus-truncated AAT (ΔAAT) was also prepared by digesting AAT with metalloproteinase. Primary CD4+ T cells were then co-cultured with HIV-1 with the presence or absence of AAT/C/ΔAAT to detect cis-infection of HIV-1. The interaction between AAT/C/ΔAAT and gp120/gp41 was also measured. Meanwhile, HIV-1 reverse transcriptase activity and viral DNA integration were also detected in these lymphocytes. The results demonstrated that AAT and C, not ΔAAT, inhibited HIV-1 entry by directly interacting with gp41. Meanwhile, AAT, C and ΔAAT could not directly interfere with the steps of viral RNA reverse transcription and viral DNA integration.
AAT inhibits HIV-1 entry by directly interacting with gp41 through its C-terminus and thereby inhibits HIV-1 infection.
Human immunodeficiency virus type 1 (HIV-1) is hard to propagate in infected and unaltered whole blood, unless the blood is diluted and active lymphocytes are present [1, 2]. Investigations of HIV-1 replication in patients reveal that HIV-1 proliferation is confined in the lymph nodes where the concentration of specific serum constituents is lower [3, 4]. These facts suggest that human body produces endogenous inhibitors for HIV and acquired immune deficiency syndrome (AIDS) progression. Further studies show that various components in human blood and tissues are possible candidates [5–7]. Among these components, serine protease inhibitors are promising. Studies report that salivary secretary leukocyte protease inhibitor suppresses HIV-1 replication [8, 9]. Researchers also focus on alpha-1-antitrypsin (AAT), a 394 amino acid, 52 kDa glycoprotein synthesized in the liver and secreted into the circulation [10–15]. AAT is consistently present in the serum of healthy individuals (1.5 ~ 3.5 mg/mL), although its concentration can increase several times upon inflammation [16, 17]. Study of a clinic case reveals that pre-existing AAT deficiency is associated with accelerated HIV/AIDS progression, which suggests that AAT might be an endogenous HIV/AIDS suppressor [13, 18].
The lifespan of HIV can be divided into two major processes, infection and replication [19, 20]. HIV-1 infection begins with the interaction between viral gp120/gp41 and CD4/co-receptors on host cells and ends with the integration of viral DNA into host genome. This process contains several middle steps, including the entry of viral core, reverse transcription of viral RNA and nuclear translocation of viral DNA [21, 22]. HIV-1 replication begins with the transcription and translation of viral genes, which lead to the package, budding and release of new virus, which is then processed to become infection-competent virus . Our previous study reveals that AAT enters the cytosol of infected CD4+ T cells through LRP1-mediated endocytosis process, where it directly interacts with nuclear factor kB (NF-kB) inhibitor (IkBα) and thereby alters its ubiquitinylation pattern to block NF-kB activation and HIV-1 replication [14, 24, 25]. Munch’s study suggests that the C-proximal fragment of AAT (A.A. 353–372), VIRIP, inhibits HIV-1 infection . However, it is unclear whether and how intact 55KD AAT interacts with gp120-covered gp41on the viral membrane to inhibit HIV-1 infection, due to its big size that might shield its VIRIP domain. It is also unknown whether AAT inhibits HIV-1 infection by targeting on single or multiple steps: viral core entry, viral RNA reverse transcription or viral DNA integration. Moreover, it is still unclear whether C-terminus of AAT is the only functional domain.
In the present study, we sought to investigate the accurate mechanism of intact AAT inhibiting HIV infection. The results demonstrate that AAT and synthesized C-terminus fragment of AAT (C), not C-terminus-truncated AAT (ΔAAT), inhibits HIV-1 entry into CD4+ T cells. The inhibitory effect is mediated through their direct interactions with gp41. Additionally, AAT, C and ΔAAT did not directly affect the steps of viral RNA reverse transcription and viral DNA integration. Thus, our results clarify how AAT inhibits HIV-1 infection in primary CD4+ T cells. Combined with the previous finding that AAT inhibits HIV-1 replication in infected cells [15, 24, 25], it is clear that AAT can suppress HIV/AIDS pathogenesis through inhibiting both HIV infection and replication in vitro, which might provide some useful information for HIV/AIDS study and drug development.
Human plasma AAT was obtained from Sigma [purity and quality were analyzed by electrophoresis and mass spectrometry following the methods described below (Additional file 1: Figure S1)]. ΔAAT were prepared by digesting AAT. Briefly, AAT and metalloproteinase from S. aureus (Sigma) were co-cultured at 40:1 (molar ratio) for 3 h at 37 °C in 50 mM NH4HCO3. Next, protease was removed by using HiTrap benzamidine column (Amersham) and then passed through 10KD MWCO spin filter (Millipore) to separate truncated AAT. The truncation of AAT was verified by electrophoresis and mass spectrometry (Additional file 1: Figure S1) and also justified by the loss of ability to bind trypsin and elastase (Sigma) [26, 27]. C-terminus of AAT (C) (A.A. 345–384: KGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIDQNTKSP) was synthesized in Genscript and the purity and accuracy of peptide was also analyzed by electrophoresis and mass spectrometry (Additional file 1: Figure S1). HIV-1 integrase inhibitor raltegravir (RAL), HIV-1 reverse transcriptase inhibitor emtricitabine (FTC) and HIV-1 entry inhibitor enfuvirtide (ENF) were from Santa Cruz Biotechnology.
HIV-1 stock was prepared by propagating HIV-1NL4.3 (X4 virus) in mitogen-stimulated peripheral blood mononuclear cells (PBMC) as before . Unless otherwise noted, HIV-1 infection was performed using HIV-1NL4.3. HIV-1 primary isolates, 91US054 (X4 virus) or 92US714 (R5 virus) (NIH AIDS Research and Reference Reagent Program), were also propagated in mitogen-stimulated PBMCs in complete culture medium (CM) [RPMI1640 with HEPES (20 mM), nonessential amino acids, L-glutamine (2 mM), 10 % heat-inactivated, defined FBS (Life Technologies/Invitrogen) and gentamicin (50 mg/ml; Life Technologies)]. Additionally, GFP-labeled HIV-1NL4.3 was made by transfecting a vector coding for HIV-1NL4.3 with GFP insertion in gag region (HIV-1 Gag-iGFP) into HEK293T cells. GFP-labeled pseudotyped HIV-1NL4.3 was made by transfecting a vector coding for HIV-1NL4.3 with GFP insertion in gag region and VSV-G replacing of Env (VSV-G-pseudotyped HIV-1 Gag-iGFP) into HEK293T cells (both vectors were generous gifts from Dr. Yuntao Wu; Dr. Wu also provided us pure gp120 and gp41). After harvesting, virus was purified, concentrated, and stored in −80 °C.
CD4+ T cell isolation and infection
PBMCs were extracted from the whole blood of healthy, HIV-negative donors using Ficoll-Paque-Plus (GE Healthcare) as directed. CD4+ T cells were then isolated from these PBMCs using a CD4+ T cell isolation Kit II (Miltenyi Biotech) as directed. Next, isolated CD4+ T cells were activated and maintained as before . To ensure that CD4- and coreceptor-dependent infection (cis-infection) is not interfered by the endocytosis of viral particle that is an alternative way of HIV infection in some cases (trans-infection) , activated CD4+ T cells were cultured for 30 min in conditioned complete medium [complete medium with the endocytosis inhibitor cocktail (5 μg/mL methyl-beta-cyclodextrin, filipin and chlorpromazine)] before and during infection, which was washed off after 2 h’ infection and maintained in normal complete medium when prolonged incubation was needed.
ELISA assay for HIV-1 p24 detection
To detect cytosol p24, CD4+ T cells were cultured with HIV-1. These cells were then suspended in Lysis Buffer [50 mM Tris–HCl (pH7.4), 1 % CHAPS, 250 mM NaCl, 0.5 % Triton X-100, 1 % Igepal CA-630, 1 mM DTT, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 4 mM EDTA, protease inhibitor cocktail (Roche)] and vortexing for 60 s. The mixture was then incubated on ice for 15 min and homogenized with a small gauge needle by drawing 3 times. After homogenizing, the mixture was centrifuged at 14,000 × g for 10 min at 4 °C to collect the supernatant (whole cell proteins containing viral proteins). HIV-1 p24 was detected using the HIV-1 p24 antigen ELISA kit (ZeptoMetrix Corporation) following the direction. For HIV-1 replication, the supernatant fluid of the culture system was collected to detect HIV-1 p24 using the HIV-1 p24 antigen ELISA kit (ZeptoMetrix Corporation) following the direction.
HIV-1 RNA detection
Supernatant with HIV-1 viral particles was collected for viral RNA quantitation. Viral RNA was isolated using QIAamp viral RNA mini Kit (Qiagen). Isolated viral RNA was reverse-transcribed into cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). Quantitative RT-PCR using TaqMan Universal PCR Master Mix (Applied Biosystems) used the following primers and probe: forward primer: 59-TGGGTACCAGCACACAAAGG-39 (nt 3696 in HXB2); reverse primer: 59-ATCACTAGCCATTGCTCTCCAAT-39 (nt 3850 in HXB2); and probe: ATTGGAGGAAATGAAC-MBG (FAM labeled) at 900 nM (primers) and 250 nM (probe). Quantitative PCR conditions were as follows: 50 °C for 2 min (1 cycle), 95 °C for 10 min (1 cycle), followed by 60 cycles of: 95 °C for 15 s and 60 °C for 1 min in an ABI 7500 thermocycler (Applied Biosystems). A standard curve was prepared using known concentrations (i.e., copy numbers) of ACH-2 DNA to determine the number of copies of viral RNA present in the cultures.
Alu-PCR assay for viral DNA integration detection
Cells were washed and resuspended in DNA-STAT 60 (Tel-Test) to extract genomic DNA following the protocol manufacture provided. Extracted DNA was then used to run Alu-gag PCR [forward primer: 5′-GCC TCC CAA AGT GCT GGG ATT ACA G-3′; reverse primer (HIV gag; nt 1505–1486): 5′-GTT CCT GCT ATG TCA CTT CC-3′]. PCR conditions were as follows: 95 °C × 2 min (1 ×), followed by 40 cycles of: 95 °C × 15 s, 50 °C × 15 s, 72 °C × 3.5 min. After Alu-gag PCR, RU5 quantitative real time PCR was carried out with the following primers and probe. R forward primer (nt 518–539): 5′-TTA AGC CTC AAT AAA GCT TGC C-3′, U5 reverse primer (nt 647–628): 5′-GTT CGG GCG CCA CTG CTA GA-3′, RU5 probe (nt 584–559): 5′-FAM-CCA GAG TCA CAC AAC AGA CGG GCA CA-MBG-3′) as directed. Meanwhile, beta-globin was also detected using the same DNA as template. Forward primer: 5′- CCC TTG GAC CCA GAG GTT CT-3′, reverse primer: 5′- CGA GCA CTT TCT TGC CAT GA-3′, probe: 5′- VIC-GCG AGC ATC TGT CCA CTC CTG ATG CTG TTA TGG GCG CTC GC-TARAMA-3′) as directed. PCR conditions were as follows: 50 °C × 2 min (1 ×), 95 °C × 20 s (1 ×); followed by 60 cycles of: 95 °C × 3 s, 60 °C × 30 s. A standard curve was prepared using known concentrations (i.e., copy numbers) of ACH-2 DNA to determine the number of copies of viral DNA integrated into the host genome. The primers were used at 900 nM and the probe at 250 nM.
Detection of HIV-1 reverse transcriptase activity
Infected CD4+ T cells were suspended lysis buffer [50 mM Tris (pH 7.4), 500 mM NaCl, 1 % Triton X-100, 2 mM PMSF, 20 % glycerol, 1 mM DTT and protease inhibitor cocktail (Roche)] and vortexed for 60 s. The mixture was incubated on ice for 45 min and homogenized with a small gauge needle by drawing 3 times. After homogenizing, the mixture was centrifuged at 14,000 × g for 10 min at 4 °C to collect the supernatant containing HIV-1 reverse transcriptase. Next, the supernatant was treated with DNase I (Ambion, Inc.) to remove contaminating DNA. The activity of HIV-1 reverse transcriptase was detected by EnzChek® Reverse Transcriptase Assay Kit (Invitrogen) following the protocol manufacture provided.
For whole cell extraction, cells were collected and washed. For viral protein extraction, HIV-1 particles were collected, washed, and concentrated by centrifuging at 100,000 × g for 2 h at 4 °C. RIPA Lysis Buffer was then added to cell or viral pellet and vortexed for 60 s. The mixture was incubated on ice for 45 min and homogenized with a small gauge needle by drawing 3 times. After homogenizing, the mixture was centrifuged at 14,000 × g for 10 min at 4 °C to collect the supernatant (whole cell proteins or viral proteins).
For membrane protein extraction, cells were washed with cold PBS and membrane proteins were extracted with membrane protein extraction kit (BioVision) following the protocol manufacture provided.
Western blot assay
Western blotting was carried out following the previous protocol . After blocking, the membrane was incubated with the primary antibody [goat anti-human gp120 (Abcam), goat anti-human gp41 (Abcam), goat anti-human AAT (Bethyl), mouse anti-human CD4 (Immunotech), mouse anti-human CCR5 (Pharmingem), mouse anti-human CXCR4 (R&D), or rabbit anti-human β-actin (Abcam)]. Next, secondary antibody [anti-goat IgG-HRP (Chemicon international), anti-mouse IgG-HRP (Chemicon international), or anti-rabbit IgG-HRP (Chemicon international)] was used to link to the primary antibody and then developed using ECL Advance Western Blotting Detection Kit (GE Healthcare).
Flow cytometry assay
Antibodies used were: CD4 allophycocyanin (APC), CXCR4 pacific blue, CCR5 fluorescein isothiocyanate (FITC) and antibody isotope controls were from BD Biosciences. Upon analysis, cells were washed and incubated for 20 min at room temperature in PBS containing 2 % BSA and antibodies or antibody isotope controls. Cells were then collected and washed twice in ice-cold PBS. The samples were analyzed on flow cytometer. For CD4+ T cell infection with HIV-1NL4.3 (HIV-1 Gag-iGFP), activated CD4+ T cells were infected with GFP-labeled HIV-1NL4.3 and unbound viruses were then removed by washing three times. GFP in CD4+ T cells was analyzed on flow cytometer to determine HIV-1 entry.
Membrane receptor interaction identification
For HIV-1 membrane receptor identification, HIV-1 was washed and concentrated by centrifuging at 100,000 × g for 2 h at 4 °C. Next, viral particles were incubated with AAT and dithiobis (succinimidylpropionate) (DSP, Thermo Scientific) was added to stabilize the interaction between AAT and HIV-1 membrane protein. Next, the reaction was stopped by adding Tris buffer and viral proteins were then extracted following the whole viral protein extraction protocol described above. For cell membrane receptor identification, cells were incubated with AAT. Excess AAT was washed off and DSP was added to stabilize the interaction between AAT and cellular membrane protein. The reaction was also stopped by adding Tris buffer and the membrane proteins were extracted with membrane protein extraction kit (BioVision) following the provided protocol described above. Isolated cell membrane proteins or viral membrane proteins were incubated with AAT-specific antibody to precipitate proteins that interacted with AAT. Precipitated proteins were separated by SDS-PAGE and specific protein bands were cut and digested with a sequencing grade modified trypsin (Promega) to identify the proteins by peptide mass fingerprinting assay using a high-performance liquid chromatography-mass spectrometry system (HPLC-MS) following the protocol described below.
In-gel digestion for peptide mass fingerprinting assay
The proteins were separated by SDS-PAGE and the appropriate bands were cut for peptide mass fingerprinting assay following our previous protocol . Digested peptides were analyzed on the HPLC-MS system (Applied Biosystem API QSTAR pulsar I LC/MS system or Thermo scientific LTQ XL Orbitrap LC/MS system). The proteins were identified by searching the specific mass spectrum in the database (Mascot).
In-gel filter-aided sample preparation (FASP) for peptide mass fingerprinting assay
Proteins were separated by SDS-PAGE and the appropriate bands were collected for FASP assay following the previous protocol . The peptides were also analyzed on Applied Biosystem API QSTAR pulsar I LC/MS system. The proteins were identified by searching the specific mass spectrum in the Mascot database.
Surface plasmon resonance (SPR) assay
AAT, ΔAAT or C-terminus fragment of AAT binding with gp41 and gp120 was detected on a BIAcore 3000 biosensor system (Pharmacia Biosensor AB) using SPR assay. Briefly, a carboxymethylated CM5 sensor chip was activated with 1:1 mixture of 0.4 M N-ethyl-N -(3-dimethylaminopropyl) carbodiimide and 0.1 M′ N-hydroxysuccinimide. AAT (0.5 g/L in 10 mM NaOAc, pH5), ΔAAT (0.47 g/L in 10 mM NaOAc, pH5) or synthesized C-terminus fragment of AAT (0.05 g/L in 10 mM NaOAc, pH5) were then immobilized on the sensor chip by amine-coupling according to the manufacturer’s instructions. Unreacted sites were blocked with 1 M ethanolamine/HCl (pH 8). Control flow cells (blank) were activated and blocked in the absence of AAT, ΔAAT or synthesized C-terminus fragment of AAT. Flow cells were routinely equilibrated with running buffer (PBS, 0.005 % surfactant P20). Analyst gp41 and gp120 were diluted in the running buffer and allowed to interact with the sensor surface by a 250-s injection. Different concentrations of gp120 and gp41 were injected, each at the flow rate of 10 μL/min at 25 °C. Data from duplicate assays were modeled for binding equilibrium.
Every experiment was repeated at least three times with different donors. Analysis of data was performed using Student t test. P values of ≤ 0.05 were considered significant. Unless specifically stated, the error bars indicate the standard errors of the means (SEM).
Studies using human cells/tissues
Studies using blood or tissue-derived cells obtained from humans were reviewed and approved by an appropriate institutional review committee.
As an alternative mechanism, AAT might directly interact with CD4, CCR5 or CXCR4, or other known HIV-1 infection-related proteins on CD4+ T cells to interfere with the interaction between HIV-1 particles and host cells. To test this postulate, CD4+ T cells were incubated with AAT or ΔAAT to precipitate the cytoplasm membrane proteins interacting with AAT or ΔAAT for protein identification by peptide mass fingerprinting assay. The results revealed that precipitated complexes contained low density lipoprotein receptor-related protein 1, ATP-binding cassette protein and solute carrier protein, which are not related to HIV-1 entry in CD4+ T cells . Meanwhile, CD4, CCR5, CXCR4 and other known HIV-1 infection-related proteins were not detected in precipitated complexes (data not shown).
The study of a clinic case reveals that pre-existing AAT deficiency is associated with accelerated HIV/AIDS progression [13, 18]. Studies reveal that AAT inhibits HIV-1 replication [10, 11, 14, 15]. Constitutive expression of AAT inhibits HIV-1 replication by blocking gp160 and p55 processing in cell lines or primary human lymphocytes . Meanwhile, AAT also inhibits HIV-1 replication by blocking the activation of NF-kB [14, 15, 24, 25, 32]. In the present study, we found that viral replication in AAT-pretreated CD4+ T cells was much lower than that in CD4+ T cells without AAT pretreatment, suggesting that AAT might exert its inhibitory effect on both HIV-1 infection and replication. However, several critical issues still need to be addressed. In the present study, our results show that AAT and C, but not ΔAAT, directly interact with gp41, which might then inhibit HIV-1 entry into CD4+ T cells. Moreover, AAT/ΔAAT/C did not directly interfere with the steps of viral RNA reverse transcription and viral DNA integration. To our surprise, the activity of HIV-1 reverse transcriptase decreased with the elongation of incubation time of the cells. This might be caused by the degradation of HIV-1 reverse transcriptase in the cytosol of CD4+ T cells, which is a general bioprocess in the cell and also leaves us a good topic to follow in the future. Therefore, these observations eliminate the concerns that 52KD AAT might be too big to directly interact with gp120-covered gp41 on the membrane of HIV-1 viral particle. The results also indicate that AAT C-terminus is the only essential functional domain to inhibit HIV-1 infection.
Normally, HIV-1 infection begins with the interaction between gp120/gp41 and CD4/co-receptors, which is then followed by viral core entry, RNA reverse transcription and DNA integration into the host genome [19, 22]. In the present study, AAT, C and ΔAAT did not directly target the steps of viral RNA transcription and viral DNA integration, which means that AAT inhibits HIV-1 infection through blocking virus entry. Although studies demonstrate that AAT inhibits HIV-1 replication in infected lymphocytes [10–15, 31], relatively fewer reports investigate whether AAT inhibits HIV-1 infection of uninfected cells and the mechanism is not fully elucidated yet. Usually, HIV-1 entry into host cells involves the interaction between viral gp120/gp41 and host CD4/CXCR4 or CCR5 . Some researchers, however, suggest that AAT inhibition of HV-1 entry is related to the interaction between AAT and host cell membrane proteins . In the present study, our results did not provide any indication to suggest that AAT interacts with host membrane proteins, thereby inhibiting HIV-1 entry. AAT also did not alter the expression of CD4, CXCR4 and CCR5. Moreover, AAT also did not directly interact with CD4, CCR5, CXCR4 and other HIV-1 infection-related proteins. In contrast, AAT directly interacted with viral gp41. These observations together suggest that intact AAT could interact with viral gp41 to interfere with the interaction between gp120/gp41 and CD4/CCR5 or/CXCR4 and thereby inhibiting HIV-1 entry, which is consistent with previous studies . Moreover, AAT without the C-terminus could not interact with gp41, which indicates that the C-terminus of AAT is an essential functional domain. Munch et al.’s study also reveals that VIRIP at the C-terminus of AAT inhibits HIV-1 infection . When ΔAAT, AAT or synthesized C-terminus fragment of AAT was cultured with gp41 or gp120, we detected direct interaction between AAT/synthesized C-terminus fragment of AAT, but not ΔAAT, and gp41. Thus, collectively with the findings of Munch’s , it is clear that the inhibitory effect of AAT on HIV-1 infection is mediated through the direct interaction between the C-terminus of AAT and gp41, which then interferes with the entry of HIV-1 into the host cells.
Studies have showed that AAT inhibits HIV-1 replication [15, 36]. Our previous study reveals that AAT enters the cytosol of infected CD4+ T cells and directly interacts with cytosolic IkBα to alter its ubiquitinylation pattern. The change of IkBα ubiquitinylation pattern results in the inhibition of NF-kB activation [15, 24]. In infected cells, NF-kB activation is critical for HIV-1 replication . In the present study, our results reveal that AAT inhibits HIV-1 infection not by directly targeting the steps of viral RNA reverse transcription and viral DNA integration. The inhibitory effect of AAT on HIV-1 infection is mediated through the direct interaction between AAT's C-terminus and gp41, which thereby inhibits HIV-1 entry into the host cells. Therefore, these results together indicate that AAT works through multiple ways to exert its inhibitory effects on HIV/AIDS pathogenesis, which may provide useful information for drug development for HIV/AIDS treatment.
AAT, alpha-1-antitrypsin; AIDS, acquired immune deficiency syndrome; APC, allophycocyanin; C, synthesized C-terminus of AAT; CM, complete culture medium; DSP, dithiobis succinimidylpropionate; ENF, HIV-1 entry inhibitor enfuvirtide; FITC, isothiocyanate; FTC, HIV-1 reverse transcriptase inhibitor emtricitabine; HIV-1, human immunodeficiency virus type 1; HPLC-MS, high-performance liquid chromatography-mass spectrometry system; IkBα, nuclear factor kB inhibitor; NF-kB, nuclear factor kB; RAL, HIV-1 integrase inhibitor raltegravir; SPR, surface plasmon resonance; ΔAAT, C terminus-truncated AAT
We acknowledged the support of Public Health Service grants from the National Institute of Allergy and Infectious Disease and the Brigham Young University grant from the College of Physical and Mathematical Sciences. We also appreciated the grants from National Natural Science Foundation of China, Science and Technology Program of Hainan Province and the Startup Fund for Distinguished Scholars of Hainan University.
This work was supported by Public Health Service grants AI91517 from the National Institute of Allergy and Infectious Disease (G.F.B.) and by a Brigham Young University Grant from the College of Physical and Mathematical Sciences (G.F.B.). This work was also partially supported by grants from National Natural Science Foundation of China (Nos. 31560021 and 31100052) (Z.L.), Science and Technology Program of Hainan Province (No. KJHZ2015-24) (Z.L.) and the Startup Fund for Distinguished Scholars of Hainan University (No. KYQD1515) (Z.L.).
Availability of data and materials
To support the findings in the present study, all data including the supplementary data are contained in the manuscript.
X.Z. designed and performed the experiments, analyzed the data and wrote the manuscript. Z.L. analyzed the data and wrote the manuscript. J.W.A. performed the experiments. G.F.B. analyzed the data and contributed the idea. J.Z. performed the experiments and analyzed the data. J.Y. analyzed the data. All authors have read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The specimens were collected under consent as part of clinical research protocol number OH99CN046, Collection and Distribution of Samples from Healthy Donors for In Vitro Research at NCI-Frederick. The protocol was reviewed and approved by the National Cancer Institute Institutional Review Board (NCI-IRB).
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- Bayliss GJ, Jesson WJ, Mortimer PP, McLean KA, Evans BA. Cultivation of human immunodeficiency virus from whole blood: effect of anticoagulant and inoculum size on virus growth. J Med Viro. 1990;31:161–4.View ArticleGoogle Scholar
- Fiore JR, Angarano G, Fico C, Monno L, Carbonara S, Salamina GF, Fracasso C, Pastore G. HIV isolation from whole blood: a new approach to HIV detection. Microbiologica. 1990;13:311–5.PubMedGoogle Scholar
- Embretson J, Zupancic M, Ribas JL, Burke A, Racz P, Tenner-Racz K, Haase AT. Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature. 1993;362:359–62.Google Scholar
- Pantaleo G, Graziosi C, Demarest JF, Butini L, Montroni M, Fox CH, Orenstein JM, Kotler DP, Fauci AS. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature. 1993;362:355–8.View ArticlePubMedGoogle Scholar
- Chiu YL, Greene WC. The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu Rev Immunol. 2008;26:317–53.View ArticlePubMedGoogle Scholar
- Bishop KN, Verma M, Kim EY, Wolinsky SM, Malim MH. APOBEC3G inhibits elongation of HIV-1 reverse transcripts. PLoS Pathog. 2008;4:e1000231.View ArticlePubMedPubMed CentralGoogle Scholar
- Venkataraman N, Cole AL, Ruchala P, Waring AJ, Lehrer RI, Stuchlik O, Pohl J, Cole AM. Reawakening retrocyclins: ancestral human defensins active against HIV-1. PLoS Biol. 2009;7:e1000095.View ArticlePubMed CentralGoogle Scholar
- McNeely TB, Dealey M, Dripps DJ, Orenstein JM, Eisenberg SP, Wahl SM. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus type 1 activity in vitro. J Clin Invest. 1995;96:456–64.View ArticlePubMedPubMed CentralGoogle Scholar
- McNeely TB, Shugars DC, Rosendahl M, Tucker C, Eisenberg SP, Wahl SM. Inhibition of human immunodeficiency virus type 1 infectivity by secretory leukocyte protease inhibitor occurs prior to viral reverse transcription. Blood. 1997;90:1141–9.PubMedGoogle Scholar
- Congote LF. The C-terminal 26-residue peptide of serpin A1 is an inhibitor of HIV-1. Biochem Biophys Res Commun. 2006;343:617–22.View ArticlePubMedGoogle Scholar
- Hayes VM, Gardiner-Garden M. Are polymorphic markers within the alpha-1-antitrypsin gene associated with risk of human immunodeficiency virus disease? J Infect Dis. 2003;188:1205–8.View ArticlePubMedGoogle Scholar
- Münch J, Ständker L, Adermann K, Schulz A, Schindler M, Chinnadurai R, Pöhlmann S, Chaipan, C, Biet T, Peters T, Meyer B, Wilhelm D, Lu H, Jing W, Jiang S, Forssmann WG, Kirchhoff F. Discovery and optimization of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide. Cell. 2007;129:263–75.View ArticlePubMedGoogle Scholar
- Potthoff AV, Münch J, Kirchhoff F, Brockmeyer NH. HIV infection in a patient with alpha-1 antitrypsin deficiency: a detrimental combination? AIDS. 2007;21:2115–6.View ArticlePubMedGoogle Scholar
- Sharpiro L, Pott GB, Ralston AH. Alpha-1-antitrypsin inhibits human immunodeficiency virus type 1. FASEB J. 2001;15:115–22.View ArticleGoogle Scholar
- Zhou X, Shapiro L, Fellingham G, Willardson BM, Burton GF. HIV replication in CD4+ T lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by alpha-1-antitrypsin through altered IkappaB(alpha) ubiquitination. J Immunol. 2011;186:3148–55.View ArticlePubMedPubMed CentralGoogle Scholar
- Blank CA, Brantley M. Clinical features and molecular characteristics of α1-antitrypsin deficiency. Ann Allergy. 1994;72:105–20.PubMedGoogle Scholar
- Voulgari F, Cummins P, Gardecki TI, Beeching NJ, Stone PC, Stuart J. Serum levels of acute phase and cardiac proteins after myocardial infarction, surgery, and infection. Br Heart J. 1982;48:352–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Bryan CL, Beard KS, Pott GB, Rahkola J, Gardner EM, Janoff EN, Shapiro L. HIV infection is associated with reduced serum alpha-1-antitrypsin concentrations. Clin Invest Med. 2010;33:E384–9.PubMedGoogle Scholar
- Chan DC, Kim PS. HIV Entry and Its Inhibition. Cell. 1998;93:681–4.View ArticlePubMedGoogle Scholar
- Imamichi T. Action of anti-HIV drugs and resistance: reverse transcriptase inhibitors and protease inhibitors. Curr Pharm Des. 2004;10:4039–53.View ArticlePubMedGoogle Scholar
- Tarrago-Litvak L, Andreola ML, Fournier M, Nevinsky GA, Parissi V, de Soultrait VR, Litvak S. Inhibitors of HIV-1 reverse transcriptase and integrase: classical and emerging therapeutical approaches. Curr Pharm Des. 2002;8:595–614.View ArticlePubMedGoogle Scholar
- Wyatt R, Sodroski J. The HIV-1 Envelope Glycoproteins: Fusogens, Antigens, and Immunogens. Science. 1998;280:1884–8.View ArticlePubMedGoogle Scholar
- Zheng YH, Lovsin N, Peterlin BM. Newly identified host factors modulate HIV replication. Immunol Lett. 2005;97:225–34.View ArticlePubMedGoogle Scholar
- Zhou X, Liu Z, Shapiro L, Yang J, Burton GF. Low-density Lipoprotein Receptor-related Protein 1 Mediates Alpha 1 Antitrypsin Internalization in CD4+ T Lymphocytes. J Leukoc Biol. 2015;98:1027–35.View ArticlePubMedGoogle Scholar
- Thacker TC, Zhou X, Estes JD, Jiang Y, Keele BF, Elton TS, Burton GF. Follicular Dendritic Cells and Human Immunodeficiency Virus Type 1 Transcription in CD4+ T Cells. J Virol. 2009;83:150–8.View ArticlePubMedGoogle Scholar
- Huang H, Campbell SC, Nelius T, Bedford DF, Veliceasa D, Bouck NP, Volpert OV. Alpha-1-antitrypsin inhibits angiogenesis and tumor growth. Int J Cancer. 2004;112:1042–8.View ArticlePubMedGoogle Scholar
- Rapala-Kozik M, Potempa J, Nelson D, Kozik A, Travis J. Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases. Biol Chem. 1999;380:1211–6.View ArticlePubMedGoogle Scholar
- Wu L, KewalRamani VN. Dendritic-cell interactions with HIV: infection and viral dissemination. Nat Rev Immunol. 2006;6:859–68.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhou X, Li J, Yang W. Calcium/calmodulin-dependent protein kinase II regulates cyclooxygenase-2 expression and prostaglandin E2 production by activating cAMP-response element-binding protein in rat peritoneal macrophages. Immunology. 2014;143:287–99.View ArticlePubMedPubMed CentralGoogle Scholar
- Hübner W, McNerney GP, Chen P, Dale BM, Gordon RE, Chuang FYS, Li XD, Asmuth DM, Huser T, Chen BK. Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses. Science. 2009;323:1743–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Cordelier P, Strayer DS. Conditional expression of alpha1-antitrypsin delivered by recombinant SV40 vectors protects lymphocytes against HIV. Gene Ther. 2003;10:2153–6.View ArticlePubMedGoogle Scholar
- Zhou X, Yang W, Li J. Ca2 + − and Protein Kinase C-dependent Signaling Pathway for Nuclear Factor-kB Activation, Inducible Nitric-oxide Synthase Expression, and Tumor Necrosis Factor-α Production in Lipopolysaccharide-stimulated Rat Peritoneal Macrophages. J Biol Chem. 2006;281:31337–47.View ArticlePubMedGoogle Scholar
- Hughes A, Nelson M. HIV entry: new insights and implications for patient management. Curr Opin Infect Dis. 2009;22:35–42.View ArticlePubMedGoogle Scholar
- Congote LF. Serpin A1 and CD91 as host instruments against HIV-1 infection: are extracellular antiviral peptides acting as intracellular messengers? Virus Res. 2007;125:119–34.View ArticlePubMedGoogle Scholar
- Kramer HB, Lavender KJ, Qin L, Stacey AR, Liu MK, di Gleria K, Gasper-Smith N, Haynes BF, McMichael AJ, Borrow P, Kessler BM. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection. PLoS Pathog. 2010;6:e1000893.Google Scholar
- Cordelier P, Zern MA, Strayer DS. HIV-1 proprotein processing as a target for gene therapy. Gene Ther. 2003;10:467–77.View ArticlePubMedGoogle Scholar
- Keele BF, Tazi L, Gartner S, Liu Y, Burgon TB, Estes JD, Thacker TC, Crandall KA, McArthur JC, Burton GF. Characterization of the follicular dendritic cell reservoir of human immunodeficiency virus type 1. J Virol. 2008;82:5548–61.View ArticlePubMedPubMed CentralGoogle Scholar