Publications by authors named "Guido Poli"

103 Publications

Biobanking for COVID-19 research.

Panminerva Med 2020 Oct 19. Epub 2020 Oct 19.

Vita-Salute San Raffaele University, Milan, Italy.

Background: Biobanks are imperative infrastructures, particularly during outbreaks, when there is an obligation to acquire and share knowledge as quick as possible to allow for implementation of science-based preventive, diagnostic, prognostic and therapeutic strategies.

Methods: We established a COVID-19 biobank with the aim of collecting high-quality and well-annotated human biospecimens, in the effort to understand the pathogenic mechanisms underlying COVID-19 and identify therapeutic targets (COVID-BioB, NCT04318366). Here we describe our experience and briefly review the characteristics of the biobanks for COVID-19 that have been so far established.

Results: A total of 46,677 samples have been collected from 913 participants (63.3% males, median [IQR] age 62.2 [51.2 - 74.0] years) since the beginning of the program. Most patients (66.9%) had been admitted to hospital for COVID-19, with a median length of stay of 15.0 (9.0 - 27.0) days. A minority of patients (13.3% of the total) had been admitted for other reasons and subsequently tested positive for SARS-CoV-2. The remainder were managed at home after being seen at the Emergency Department.

Conclusions: Having a solid research infrastructure already in place, along with flexibility and adaptability to new requirements, allowed for the quick building of a COVID-19 biobank that will help expand and share the knowledge of SARS-CoV-2.
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http://dx.doi.org/10.23736/S0031-0808.20.04168-3DOI Listing
October 2020

HLA-E-restricted CD8 T Lymphocytes Efficiently Control and HIV-1 Coinfection.

Am J Respir Cell Mol Biol 2020 04;62(4):430-439

Central Laboratory for Advanced Diagnosis and Biomedical Research.

We investigated the contribution of human leukocyte antigen A2 (HLA-A2) and HLA-E-restricted CD8 T cells in patients with and human immunodeficiency virus 1 (HIV-1) coinfection. HIV-1 downregulates HLA-A, -B, and -C molecules in infected cells, thus influencing recognition by HLA class I-restricted CD8 T cells but not by HLA-E-restricted CD8 T cells, owing to the inability of the virus to downmodulate their expression. Therefore, antigen-specific HLA-E-restricted CD8 T cells could play a protective role in and HIV-1 coinfection. HLA-E- and HLA-A2-restricted -specific CD8 T cells were tested for cytotoxic and microbicidal activities, and their frequencies and phenotypes were evaluated in patients with active tuberculosis and concomitant HIV-1 infection. HIV-1 and coinfection caused downmodulation of HLA-A2 expression in human monocyte-derived macrophages associated with resistance to lysis by HLA-A2-restricted CD8 T cells and failure to restrict the growth of intracellular . Conversely, HLA-E surface expression and HLA-E-restricted cytolytic and microbicidal CD8 responses were not affected. HLA-E-restricted and -specific CD8 T cells were expanded in the circulation of patients with /HIV-1 coinfection, as measured by tetramer staining, but displayed a terminally differentiated and exhausted phenotype that was rescued by anti-PD-1 (programmed cell death protein 1) monoclonal antibody. Together, these results indicate that HLA-E-restricted and -specific CD8 T cells in patients with /HIV-1 coinfection have an exhausted phenotype and fail to expand in response to antigen stimulation, which can be restored by blocking the PD-1 pathway using the specific monoclonal antibody nivolumab.
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http://dx.doi.org/10.1165/rcmb.2019-0261OCDOI Listing
April 2020

Interferon-inducible TRIM22 contributes to maintenance of HIV-1 proviral latency in T cell lines.

Virus Res 2019 08 25;269:197631. Epub 2019 May 25.

Unit of Viral Pathogens and Biosafety, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy. Electronic address:

The human immunodeficiency virus type-1 (HIV-1) establishes a state of latent infection in a small number of CD4 T lymphocytes that, nonetheless, represent a major obstacle to viral eradication. We here show that Tripartite Motif-containing protein 22 (TRIM22), an epigenetic inhibitor of Specificity protein 1 (Sp1)-dependent HIV-1 transcription, is a relevant factor in maintaining a state of repressed HIV-1 expression at least in CD4 T cell lines. By knocking-down (KD) TRIM22 expression, we observed an accelerated reactivation of a doxycycline (Dox)-controlled HIV-1 replication in the T lymphocytic SupT1 cell line. Furthermore, we here report for the first time that TRIM22 is a crucial factor for maintaining a state of HIV-1 quiescence in chronically infected ACH2 -T cell line while its KD potentiated HIV-1 expression in both ACH-2 and J-Lat 10.6 cell lines upon cell stimulation with either tumor necrosis factor-α (TNF-α) or histone deacetylase inhibitors (HDACi). In conclusion, TRIM22 is a novel determinant of HIV-1 latency, at least in T cell lines, thus representing a potential pharmacological target for strategies aiming at curtailing or silencing the pool of latently infected CD4 T lymphocytes constituting the HIV-1 reservoir in individuals receiving combination antiretroviral therapy.
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http://dx.doi.org/10.1016/j.virusres.2019.05.009DOI Listing
August 2019

The ATP/P2X7 axis in human immunodeficiency virus infection of macrophages.

Curr Opin Pharmacol 2019 08 20;47:46-52. Epub 2019 Mar 20.

Viral Pathogens and Biosafety Unit, San Raffaele Scientific Institute, Milano, Italy; Vita-Salute San Raffaele University School of Medicine, Milano, Italy. Electronic address:

HIV-1 infects CD4+ T lymphocytes with a 'helper' function and myeloid cells, mostly tissue-resident macrophages. While infection of CD4 T lymphocytes in the absence of combination antiretroviral therapy (cART) leads to their depletion and to a profound immunodeficiency, macrophages are resistant to virus-induced cytopathicity and are a source of infectious virus, particularly in the central nervous system (CNS). Infected macrophages are characterized by accumulating newly formed viral particles (virions) in subcellular vacuoles defined as 'virus-containing compartments (VCC)', derived from invaginations of the plasma membrane, that are poorly accessible to antiretroviral agents and anti-HIV antibodies. Several factors favor the accumulation of HIV-1 virions in VCC in vitro, whereas extracellular ATP, via binding to its receptor P2X7, is the only agent described thus far as capable of triggering the rapid release of VCC-sequestered virions without simultaneously causing the death of infected macrophages. Thus, the eATP/P2X7 axis could be exploited to achieve a pharmacological control of VCC-associated viral reservoir in individuals under effective cART.
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http://dx.doi.org/10.1016/j.coph.2019.02.006DOI Listing
August 2019

Reversible Human Immunodeficiency Virus Type-1 Latency in Primary Human Monocyte-Derived Macrophages Induced by Sustained M1 Polarization.

Sci Rep 2018 09 24;8(1):14249. Epub 2018 Sep 24.

Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy.

We have reported that short-term stimulation of primary human monocyte-derived macrophages (MDM) with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), i.e. M1 polarization, leads to a significant containment of virus replication. Here we show that M1-MDM restimulation with these cytokines 7 days after infection (M1 MDM) promoted an increased restriction of HIV-1 replication characterized by very low levels of virus production near to undetectable levels. In comparison to control and M1-MDM that were not restimulated, M1 MDM showed a stronger reduction of both total and integrated HIV DNA as well as of viral mRNA expression. M1 MDM were characterized by an upregulated expression of restriction factors acting at the level of reverse transcription (RT), including apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) and APOBEC3G, but not SAM domain and HD domain-containing protein 1 (SAMHD1). M1 MDM also showed an increased expression of Class II Transactivator (CIITA) and Tripartite Motif22 (TRIM22), two negative regulators of proviral transcription, whereas expression and phosphorylation of transcriptional inducers of HIV-1, such as nuclear factor kB (NF-kB) and signal transducer and activator of transcription 1 (STAT1), were not impaired in these cells. The almost quiescent state of the infection in M1 MDM was promptly reversed by coculture with mitogen-stimulated leukocytes or cell incubation with their filtered culture supernatant. M1 MDM harbored replication-competent HIV-1 as virus spreading following cell stimulation was fully prevented by the RT inhibitor lamivudine/3TC. Selective reactivation of proviral expression in M1 MDM, but not in control or in M1-MDM that were not restimulated, was confirmed in cells infected with single round Vesicular Stomatitis Virus-G-pseudotyped HIV-1. Thus, M1 MDM represent an in vitro model of reversible, almost quiescent HIV-1 infection of primary human macrophages that could be further exploited for "Cure" related investigations.
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http://dx.doi.org/10.1038/s41598-018-32451-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155284PMC
September 2018

Highlights from the 8 International Workshop on HIV Persistence during Therapy, 12-15 December 2017, Miami, FL, USA.

J Virus Erad 2018 Apr 1;4(2):132-142. Epub 2018 Apr 1.

McGill University, Montréal, Canada.

Over 4 days, more than 500 scientists involved in HIV persistence research shared their new unpublished data and designed future perspectives towards ART-free HIV remission. This followed the format of past conferences but further focused on encouraging participation of young investigators, especially through submission of oral and poster presentations. The topic of the workshop was HIV persistence. Consequently, issues of HIV reservoirs and HIV cure were also addressed. In this article, we report the discussions as closely as possible; however, all the workshop abstracts can be found online at www.viruseradication.com.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892681PMC
April 2018

The interferon-stimulated gene TRIM22: A double-edged sword in HIV-1 infection.

Cytokine Growth Factor Rev 2018 04 10;40:40-47. Epub 2018 Feb 10.

AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, School of Medicine, Milan, Italy.

Infection of target cells by the human immunodeficiency virus type-1 (HIV-1) is hampered by constitutively expressed host cell proteins preventing or curtailing virus replication and therefore defined as "restriction factors". Among them, members of the tripartite motif (TRIM) family have emerged as important players endowed with both antiviral effects and modulatory capacity of the innate immune response. TRIM5α and TRIM19 (i.e. promyelocytic leukemia, PML) are among the best-characterized family members; however, in this review we will focus on the potential role of another family member, i.e. TRIM22, a factor strongly induced by interferon stimulation, in HIV infection in vivo and in vitro in the context of its broader antiviral effects. We will also focus on the potential role of TRIM22 in HIV-1-infected individuals speculating on its dual role in controlling virus replication and more complex role in chronic infection. At the molecular levels, we will review the evidence in favor of a relevant role of TRIM22 as epigenetic inhibitor of HIV-1 transcription acting by preventing the binding of the host cell transcription factor Sp1 to the viral promoter. These evidences suggest that TRIM22 should be considered a potential new player in either the establishment or maintenance of HIV-1 reservoirs of latently infected cells unaffected by combination antiretroviral therapy.
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http://dx.doi.org/10.1016/j.cytogfr.2018.02.001DOI Listing
April 2018

Chronically infected T-cell lines become handy for a novel assay measuring the reservoir of replication-competent HIV-1.

AIDS 2017 Nov;31(18):2555-2556

aDivision of Immunology, Transplantation, and Infectious Diseases, San Raffaele Scientific Institute bSchool of Medicine, Vita-Salute San Raffaele University, Milan, Italy.

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http://dx.doi.org/10.1097/QAD.0000000000001655DOI Listing
November 2017

HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells.

Nat Commun 2017 09 8;8(1):498. Epub 2017 Sep 8.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS, San Raffaele Scientific Institute, Milan, 20132, Italy.

HIV-1 insertions targeting BACH2 or MLK2 are enriched and persist for decades in hematopoietic cells from patients under combination antiretroviral therapy. However, it is unclear how these insertions provide such selective advantage to infected cell clones. Here, we show that in 30/87 (34%) patients under combination antiretroviral therapy, BACH2, and STAT5B are activated by insertions triggering the formation of mRNAs that contain viral sequences fused by splicing to their first protein-coding exon. These chimeric mRNAs, predicted to express full-length proteins, are enriched in T regulatory and T central memory cells, but not in other T lymphocyte subsets or monocytes. Overexpression of BACH2 or STAT5B in primary T regulatory cells increases their proliferation and survival without compromising their function. Hence, we provide evidence that HIV-1-mediated insertional activation of BACH2 and STAT5B favor the persistence of a viral reservoir in T regulatory cells in patients under combination antiretroviral therapy.HIV insertions in hematopoietic cells are enriched in BACH2 or MLK2 genes, but the selective advantages conferred are unknown. Here, the authors show that BACH2 and additionally STAT5B are activated by viral insertions, generating chimeric mRNAs specifically enriched in T regulatory cells favoring their persistence.
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http://dx.doi.org/10.1038/s41467-017-00609-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591266PMC
September 2017

Tripartite Motif-Containing Protein 22 Interacts with Class II Transactivator and Orchestrates Its Recruitment in Nuclear Bodies Containing TRIM19/PML and Cyclin T1.

Front Immunol 2017 15;8:564. Epub 2017 May 15.

Laboratory of General Pathology and Immunology, Department of Medicine and Surgery, University of Insubria, Varese, Italy.

Among interferon (IFN) inducible antiviral factors both tripartite motif-containing protein 22 (TRIM22) and class II transactivator (CIITA) share the capacity of repressing human immunodeficiency virus type 1 (HIV-1) proviral transcription. TRIM22 is constitutively expressed in a subset of U937 cell clones poorly permissive to HIV-1 replication, whereas CIITA has been shown to inhibit virus multiplication in both T lymphocytic and myeloid cells, including poorly HIV-1 permissive U937 cells, by suppressing Tat-mediated transactivation of HIV-1 transcription. Therefore, we tested whether TRIM22 and CIITA could form a nuclear complex potentially endowed with HIV-1 repressive functions. Indeed, we observed that TRIM22, independent of its E3 ubiquitin ligase domain, interacts with CIITA and promotes its recruitment into nuclear bodies. Importantly, TRIM19/promyelocytic leukemia (PML) protein, another repressor of HIV-1 transcription also acting before proviral integration, colocalize in these nuclear bodies upon TRIM22 expression induced by IFN-γ. Finally, tTRIM22 nuclear bodies also contained CyclinT1, a crucial elongation factor of HIV-1 primary transcripts. These findings show that TRIM22 nuclear bodies are a site of recruitment of factors crucial for the regulation of HIV-1 transcription and highlight the potential existence of a concerted action between TRIM22, CIITA, and TRIM19/PML to maintain a state of proviral latency, at least in myeloid cells.
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http://dx.doi.org/10.3389/fimmu.2017.00564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430032PMC
May 2017

5-Hydroxytyrosol inhibits HIV-1 replication in primary cells of the lower and upper female reproductive tract.

Antiviral Res 2017 06 7;142:16-20. Epub 2017 Mar 7.

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milano, Italy; Vita-Salute San Raffaele University, School of Medicine, Via Olgettina 58, 20132, Milano, Italy. Electronic address:

We investigated the potential anti-HIV-1 activity of the candidate microbicide 5-hydroxytyrosol (5-HT) both in primary human cervical tissue explants (CTE), established from tissues of women undergoing histerectomy, and in endometrium-associated leukocytes (EAL). CTE were exposed to either the laboratory-adapted HIV-1 or to primary viral isolates in the presence or absence of 5-HT or 3TC/lamivudine as control and were then monitored for 12 days in terms of HIV-1 p24 Gag antigen production in culture supernatants. HIV-1 replication was also evaluated in EAL by reverse transcriptase (RT) activity. The highest nontoxic concentrations of 5-HT (200 and 100 μM for CTE and EAL, respectively) exerted a significant inhibitory effect on virus replication in both primary cell systems. 5-HT did not cause significant alterations of the activation profile of CD4 and CD8 T cells, in terms of CD4, CCR5, CD25, CD69 and HLA-DR expression, although it decreased the percentage of CD38CD8 T cells. Thus, 5-HT deserves consideration as a potential candidate microbicide for preventing HIV-1 transmission or curtailing its replication in the female reproductive tract.
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http://dx.doi.org/10.1016/j.antiviral.2017.03.003DOI Listing
June 2017

Human Endometrial Stromal Cells Are Highly Permissive To Productive Infection by Zika Virus.

Sci Rep 2017 03 10;7:44286. Epub 2017 Mar 10.

Viral Pathogens and Biosafety Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

Zika virus (ZIKV) is a recently re-emerged flavivirus transmitted to humans by mosquito bites but also from mother to fetus and by sexual intercourse. We here show that primary human endometrial stromal cells (HESC) are highly permissive to ZIKV infection and support its in vitro replication. ZIKV envelope expression was detected in the endoplasmic reticulum whereas double-stranded viral RNA colocalized with vimentin filaments to the perinuclear region. ZIKV productive infection also occurred in the human T-HESC cell line together with the induction of interferon-β (IFN-β) and of IFN-stimulated genes. Notably, in vitro decidualization of T-HESC with cyclic AMP and progesterone upregulated the cell surface expression of the ZIKV entry co-receptor AXL and boosted ZIKV replication by ca. 100-fold. Thus, endometrial stromal cells, particularly if decidualized, likely represent a crucial cell target of ZIKV reaching them, either via the uterine vasculature in the viremic phase of the infection or by sexual viral transmission, and a potential source of virus spreading to placental trophoblasts during pregnancy.
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http://dx.doi.org/10.1038/srep44286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345097PMC
March 2017

Activating Killer Immunoglobulin Receptors and HLA-C: a successful combination providing HIV-1 control.

Sci Rep 2017 02 13;7:42470. Epub 2017 Feb 13.

IRCCS Giannina Gaslini, Genoa, Italy.

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.
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http://dx.doi.org/10.1038/srep42470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304173PMC
February 2017

Plastic restriction of HIV-1 replication in human macrophages derived from M1/M2 polarized monocytes.

J Leukoc Biol 2016 11 30;100(5):1147-1153. Epub 2016 Jun 30.

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy;

M1/M2 cytokine-dependent polarization of primary human MDMs has been shown to contain CCR5-dependent (R5) HIV-1 replication. In this study, a similar effect was achieved when monocytes were first polarized toward M1 or M2 and were infected 7 d after their differentiation into MDMs, regardless of whether the cytokines were removed 18 h after cell stimulation or were left in culture. Unlike polarized MDMs, no significant down-regulation of CD4 from the cell surface was observed in MDMs derived from M1/M2-polarized monocytes. A second stimulation of MDMs differentiated from M1/M2 monocytes with the opposite polarizing cytokines converted the virus replication profile according to the new stimuli. The expression of M1 and M2 markers (i.e., APOBEC3A and DC-SIGN, respectively) was induced by MDM stimulation with the opposite cytokines, although it also persisted in cells according to their first stimulatory condition. Thus, stimulation of monocytes with M1- and M2-inducing cytokines leads to a restriction of HIV-1 replication when these cells are infected several days later as differentiated MDMs. These observations imply that activation of circulating monocytes significantly influences their capacity to either support or restrict HIV-1 replication, once extravasated, and eventually to become infected as tissue macrophages.
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http://dx.doi.org/10.1189/jlb.4AB0316-158RDOI Listing
November 2016

Highlights from the Seventh International Workshop on HIV Persistence during Therapy, 8-11 December 2015, Miami, Florida, USA.

J Virus Erad 2016 Jan 1;2(1):57-65. Epub 2016 Jan 1.

General Hospital , Toulon , France.

Over 4 days, more than 270 scientists involved in HIV persistence research convened to share their data and discuss future avenues to control HIV without continuous antiretroviral therapy. This 7(th) International Workshop on HIV Persistence followed the format of the preceding conferences but more time was given for discussing abstracts submitted by the participants and selected by the Steering and Scientific Committees. The topic of the workshop is HIV persistence: consequently, issues of HIV reservoirs and HIV cure are also addressed. In this article we report as closely as possible what was discussed. However, owing to length constraints, not everything is reported here but all the Workshop abstracts can be found online (www.viruseradication.com).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946700PMC
January 2016

Zika Virus: a re-emerging pathogen with rapidly evolving public health implications.

New Microbiol 2016 Apr;39(2):86-90

Viral Pathogens and Biosafety Unit.

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April 2016

The MHC-II transactivator CIITA inhibits Tat function and HIV-1 replication in human myeloid cells.

J Transl Med 2016 Apr 18;14:94. Epub 2016 Apr 18.

Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.

Background: We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells.

Methods: U937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants.

Results: CIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.

Conclusions: U937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1 replication. The generation of Plus-CIITA cells was instrumental to demonstrate the specific contribution of CIITA in terms of inhibition of Tat activity and HIV-1 restriction, independently from other cellular factors, including TRIM22. Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.
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http://dx.doi.org/10.1186/s12967-016-0853-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835826PMC
April 2016

Immuno-Pharmacological Targeting of Virus-Containing Compartments in HIV-1-Infected Macrophages.

Trends Microbiol 2016 07 21;24(7):558-567. Epub 2016 Mar 21.

AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milano, Italy; Vita-Salute San Raffaele University, School of Medicine, Milano, Italy; Institute of Human Virology, University of Maryland, Baltimore, MD, USA. Electronic address:

In addition to CD4 T lymphocytes, HIV-1 infects tissue macrophages that can actively accumulate infectious virions in vacuolar subcellular structures mostly connected to the plasma membrane and recently termed virus-containing compartments (VCCs). The VCC-associated HIV-1 reservoir of infected macrophages can be either increased or depleted by immunologic and pharmacologic agents, at least in vitro, thus suggesting that these factors (or related molecules) could be effective in curtailing the macrophage-associated HIV-1 reservoir in infected individuals receiving combination antiretroviral therapy (cART). Here we review evidence on the pathogenic role of tissue macrophages as long-term viral reservoirs in vivo and upon in vitro infection with a particular emphasis on the immuno-pharmacological modulation of VCCs.
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http://dx.doi.org/10.1016/j.tim.2016.02.018DOI Listing
July 2016

Polymorphisms of large effect explain the majority of the host genetic contribution to variation of HIV-1 virus load.

Proc Natl Acad Sci U S A 2015 Nov 9;112(47):14658-63. Epub 2015 Nov 9.

Institute for Genomic Medicine, Columbia University, New York, NY 10032;

Previous genome-wide association studies (GWAS) of HIV-1-infected populations have been underpowered to detect common variants with moderate impact on disease outcome and have not assessed the phenotypic variance explained by genome-wide additive effects. By combining the majority of available genome-wide genotyping data in HIV-infected populations, we tested for association between ∼8 million variants and viral load (HIV RNA copies per milliliter of plasma) in 6,315 individuals of European ancestry. The strongest signal of association was observed in the HLA class I region that was fully explained by independent effects mapping to five variable amino acid positions in the peptide binding grooves of the HLA-B and HLA-A proteins. We observed a second genome-wide significant association signal in the chemokine (C-C motif) receptor (CCR) gene cluster on chromosome 3. Conditional analysis showed that this signal could not be fully attributed to the known protective CCR5Δ32 allele and the risk P1 haplotype, suggesting further causal variants in this region. Heritability analysis demonstrated that common human genetic variation-mostly in the HLA and CCR5 regions-explains 25% of the variability in viral load. This study suggests that analyses in non-European populations and of variant classes not assessed by GWAS should be priorities for the field going forward.
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http://dx.doi.org/10.1073/pnas.1514867112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664299PMC
November 2015

CD14(+) macrophages that accumulate in the colon of African AIDS patients express pro-inflammatory cytokines and are responsive to lipopolysaccharide.

BMC Infect Dis 2015 Oct 17;15:430. Epub 2015 Oct 17.

MRC Unit for Inflammation and Immunity, Department of Immunology and the Tshwane Academic Division of the National Health Laboratory Service, University of Pretoria, Pretoria, South Africa.

Background: Intestinal macrophages are key regulators of inflammatory responses to the gut microbiome and play a central role in maintaining tissue homeostasis and epithelial integrity. However, little is known about the role of these cells in HIV infection, a disease fuelled by intestinal inflammation, a loss of epithelial barrier function and increased microbial translocation (MT).

Methods: Phenotypic and functional characterization of intestinal macrophages was performed for 23 African AIDS patients with chronic diarrhea and/or weight loss and 11 HIV-negative Africans with and without inflammatory bowel disease (IBD). AIDS patients were treated with cotrimoxazole for the prevention of opportunistic infections (OIs). Macrophage phenotype was assessed by flow cytometry and immuno-histochemistry (IHC); production of proinflammatory mediators by IHC and Qiagen PCR Arrays; in vitro secretion of cytokines by the Bio-Plex Suspension Array System. Statistical analyses were performed using Spearman's correlation and Wilcoxon matched-pair tests. Results between groups were analyzed using the Kruskal-Wallis with Dunn's post-test and the Mann-Whitney U tests.

Results: None of the study participants had evidence of enteric co-infections as assessed by stool analysis and histology. Compared to healthy HIV-negative controls, the colon of AIDS patients was highly inflamed with increased infiltration of inflammatory cells and increased mRNA expression of proinflammatory cytokine (tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IFN-γ, and IL-18), chemokines (chemokine (C-C motif) ligand (CCL)2 and chemokine (C-X-C) motif ligand (CXCL)10) and transcription factors (TNF receptor-associated factor (TRAF)6 and T-box (TXB)21). IHC revealed significant co-localization of TNF-α and IL-1β with CD68(+) cells. As in IBD, HIV was associated with a marked increase in macrophages expressing innate response receptors including CD14, the co-receptor for lipopolysaccharide (LPS). The frequency of CD14(+) macrophages correlated positively with plasma LPS, a marker of MT. Total unfractionated mucosal mononuclear cells (MMC) isolated from the colon of AIDS patients, but not MMC depleted of CD14(+) cells, secreted increased levels of proinflammatory cytokines ex vivo in response to LPS.

Conclusions: Intestinal macrophages, in the absence of overt OIs, play an important role in driving persistent inflammation in HIV patients with late-stage disease and diarrhea. These results suggest intensified treatment strategies that target inflammatory processes in intestinal macrophages may be highly beneficial in restoring the epithelial barrier and limiting MT in HIV-infected patients.
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http://dx.doi.org/10.1186/s12879-015-1176-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609115PMC
October 2015

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

Proc Natl Acad Sci U S A 2015 Jun 8;112(25):E3265-73. Epub 2015 Jun 8.

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milano, Italy; Vita-Salute San Raffaele University, 20132 Milano, Italy; Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201

HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.
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http://dx.doi.org/10.1073/pnas.1500656112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485148PMC
June 2015

Spontaneous control of HIV-1 viremia in a subject with protective HLA-B plus HLA-C alleles and HLA-C associated single nucleotide polymorphisms.

J Transl Med 2014 Dec 5;12:335. Epub 2014 Dec 5.

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Disease, San Raffaele Scientific Institute, Via Olgettina n. 58, Milan, 20132, Italy.

Introduction: Understanding the mechanisms by which some individuals are able to naturally control HIV-1 infection is an important goal of AIDS research. We here describe the case of an HIV-1(+) woman, CASE1, who has spontaneously controlled her viremia for the last 14 of her 20 years of infection.

Methods: CASE1 has been clinically monitored since 1993. Detailed immunological, virological and histological analyses were performed on samples obtained between 2009 and 2011.

Results: As for other Elite Controllers, CASE1 is characterized by low to undetectable levels of plasma HIV-1 RNA, peripheral blood mononuclear cell (PBMC) associated HIV-1 DNA and reduced in vitro susceptibility of target cells to HIV-1 infection. Furthermore, a slow rate of virus evolution was demonstrated in spite the lack of assumption of any antiretroviral agent. CASE1 failed to transmit HIV-1 to either her sexual male partner or to her child born by vaginal delivery. Normal values and ratios of T and B cells were observed, along with normal histology of the intestinal mucosa. Attempts to isolate HIV-1 from her PBMC and gut-derived cells were unsuccessful, despite expression of normal cell surface levels of CD4, CCRC5 and CXCR4. CASE1 did not produce detectable anti-HIV neutralizing antibodies in her serum or genital mucosal fluid although she displayed potent T cell responses against HIV-1 Gag and Nef. CASE1 also possessed multiple genetic polymorphisms, including HLA alleles (B*14, B*57, C*06 and C*08.02) and HLA-C single nucleotide polymorphisms (SNPs, rs9264942 C/C and rs67384697 del/del), that have been previously individually associated with spontaneous control of plasma viremia, maintenance of high CD4(+) T cell counts and delayed disease progression.

Conclusions: CASE1 has controlled her HIV-1 viremia below the limit of detection in the absence of antiretroviral therapy for more than 14 years and has not shown any sign of immunologic deterioration or disease progression. Co-expression of multiple protective HLA alleles, HLA-C SNPs and strong T cell responses against HIV-1 proteins are the most likely explanation of this very benign case of spontaneous control of HIV-1 disease progression.
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http://dx.doi.org/10.1186/s12967-014-0335-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272524PMC
December 2014

Cell-to-cell vs. cell-free HIV-1 transmission from macrophages to CD4+ T lymphocytes: lessons from the virology textbook.

Authors:
Guido Poli

AIDS 2013 Sep;27(14):2307-8

AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute and Vita-Salute San Raffaele University, Milano, Italy.

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http://dx.doi.org/10.1097/QAD.0b013e328363619aDOI Listing
September 2013

Association study of common genetic variants and HIV-1 acquisition in 6,300 infected cases and 7,200 controls.

PLoS Pathog 2013 25;9(7):e1003515. Epub 2013 Jul 25.

School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

Multiple genome-wide association studies (GWAS) have been performed in HIV-1 infected individuals, identifying common genetic influences on viral control and disease course. Similarly, common genetic correlates of acquisition of HIV-1 after exposure have been interrogated using GWAS, although in generally small samples. Under the auspices of the International Collaboration for the Genomics of HIV, we have combined the genome-wide single nucleotide polymorphism (SNP) data collected by 25 cohorts, studies, or institutions on HIV-1 infected individuals and compared them to carefully matched population-level data sets (a list of all collaborators appears in Note S1 in Text S1). After imputation using the 1,000 Genomes Project reference panel, we tested approximately 8 million common DNA variants (SNPs and indels) for association with HIV-1 acquisition in 6,334 infected patients and 7,247 population samples of European ancestry. Initial association testing identified the SNP rs4418214, the C allele of which is known to tag the HLA-B*57:01 and B*27:05 alleles, as genome-wide significant (p = 3.6 × 10⁻¹¹). However, restricting analysis to individuals with a known date of seroconversion suggested that this association was due to the frailty bias in studies of lethal diseases. Further analyses including testing recessive genetic models, testing for bulk effects of non-genome-wide significant variants, stratifying by sexual or parenteral transmission risk and testing previously reported associations showed no evidence for genetic influence on HIV-1 acquisition (with the exception of CCR5Δ32 homozygosity). Thus, these data suggest that genetic influences on HIV acquisition are either rare or have smaller effects than can be detected by this sample size.
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http://dx.doi.org/10.1371/journal.ppat.1003515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723635PMC
February 2014

HIV-1 infected lymphoid organs upregulate expression and release of the cleaved form of uPAR that modulates chemotaxis and virus expression.

PLoS One 2013 29;8(7):e70606. Epub 2013 Jul 29.

Pathology Unit, "Luigi Sacco" Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy.

Cell-associated receptor for urokinase plasminogen activator (uPAR) is released as both full-length soluble uPAR (suPAR) and cleaved (c-suPAR) form that maintain ability to bind to integrins and other receptors, thus triggering and modulating cell signaling responses. Concerning HIV-1 infection, plasma levels of suPAR have been correlated with the severity of disease, levels of immune activation and ineffective immune recovery also in individuals receiving combination anti-retroviral therapy (cART). However, it is unknown whether and which suPAR forms might contribute to HIV-1 induced pathogenesis and to the related state of immune activation. In this regard, lymphoid organs represent an import site of chronic immune activation and virus persistence even in individuals receiving cART. Lymphoid organs of HIV-1(+) individuals showed an enhanced number of follicular dendritic cells, macrophages and endothelial cells expressing the cell-associated uPAR in comparison to those of uninfected individuals. In order to investigate the potential role of suPAR forms in HIV-1 infection of secondary lymphoid organs, tonsil histocultures were established from HIV-1 seronegative individuals and infected ex vivo with CCR5- and CXCR4-dependent HIV-1 strains. The levels of suPAR and c-suPAR were significantly increased in HIV-infected tonsil histocultures supernatants in comparison to autologous uninfected histocultures. Supernatants from infected and uninfected cultures before and after immunodepletion of suPAR forms were incubated with the chronically infected promonocytic U1 cell line characterized by a state of proviral latency in unstimulated conditions. In the contest of HIV-conditioned supernatants we established that c-suPAR, but not suPAR, inhibited chemotaxis and induced virus expression in U1 cells. In conclusion, lymphoid organs are an important site of production and release of both suPAR and c-suPAR, this latter form being endowed with the capacity of inhibiting chemotaxis and inducing HIV-1 expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070606PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726662PMC
August 2014

Identification of TRIM22 single nucleotide polymorphisms associated with loss of inhibition of HIV-1 transcription and advanced HIV-1 disease.

AIDS 2013 Sep;27(15):2335-44

aViral Pathogens and Biosafety Unit, Division of Immunology, Transplantation and Infectious Diseases bDepartment of Infectious Diseases cDivision of Genetics and Cell Biology, San Raffaele Scientific Institute dUniversity of Milan eGEMIB srl, Parma fAIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute gUniversità Vita-Salute San Raffaele, School of Medicine, Milan, Italy.

Objective(s): Tripartite motif-containing 22 (TRIM22) is an interferon-induced protein that inhibits HIV-1 transcription and replication in vitro. Two single nucleotide missense polymorphisms rs7935564A/G (SNP-1) and rs1063303C/G (SNP-2) characterize the coding sequence of human TRIM22 gene. We tested whether these variants affected the inhibitory effect of TRIM22 on HIV-1 replication and transcription and their potential association with HIV-1 disease.

Design: The allelic discrimination was determined in 182 HIV-1-negative and among HIV-1-positive individuals with advanced disease progression (advanced progressors; n = 57), normal progressors (n = 76), and long-term nonprogressors (LTNPs; n = 95).

Methods: Renilla luciferase activity was measured after infection of activated peripheral blood mononuclear cells (PBMCs) from an additional group of 61 blood donors with a recombinant HIV-1. HIV-1-long terminal repeat (LTR)-driven luciferase activity was tested in the presence of plasmid expressing TRIM22 variants in 293T cells. The SNP genotyping was determined by TaqMan assay.

Results: HIV-1 replication was more efficient in PBMCs from donors with SNP-1G and SNP-2G than from those with SNP-1A and SNP-2C alleles. Consistently, TRIM22-GG enhanced, whereas TRIM22-AC restricted basal HIV-1 LTR-driven transcription. In vivo, SNP-1G homozygotes and A/G heterozygotes were more frequent in advanced progressors than in LTNPs [odds ratio (OR) = 2.072, P = 0.005] or in normal progressors (OR = 1.809, P = 0.022); in contrast, SNP-2 was not associated with any state of HIV-1 disease progression. Although SNP-2 distribution was similar among the groups, TRIM22-GG haplotype was found more frequently in advanced progressors than in LTNPs (P = 0.02).

Conclusion: TRIM22 genetic diversity affects HIV-1 replication in vitro and it is a potentially novel determinant of HIV-1 disease severity.
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http://dx.doi.org/10.1097/01.aids.0000432474.76873.5fDOI Listing
September 2013

Impaired CD4+ T-cell restoration in the small versus large intestine of HIV-1-positive South Africans receiving combination antiretroviral therapy.

J Infect Dis 2013 Oct 6;208(7):1113-22. Epub 2013 Jun 6.

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston MA 02215, USA.

Background: Human immunodeficiency virus type 1 (HIV-1) infection is associated with a massive depletion of intestinal CD4(+) T cells that is only partially reversed by combination antiretroviral therapy (cART). Here, we assessed the ability of nucleoside reverse-transcriptase inhibitor/nonnucleoside reverse-transcriptase inhibitor treatment to restore the CD4(+) T-cell populations in the intestine of South African patients with AIDS.

Methods: Thirty-eight patients with advanced HIV-1 infection who had chronic diarrhea (duration, >4 weeks) and/or unintentional weight loss (>10% decrease from baseline) of uncertain etiology were enrolled. Blood specimens were collected monthly, and gastrointestinal tract biopsy specimens were collected before cART initiation (from the duodenum, jejunum, ileum, and colon), 3 months after cART initiation (from the duodenum), and 6 months after cART initiation (from the duodenum and colon). CD4(+), CD8(+), and CD38(+)CD8(+) T cells were quantified by flow cytometry and immunohistochemistry analyses, and the HIV-1 RNA load was determined by the Nuclisens assay.

Results: CD4(+) T-cell and HIV-1 RNA levels were significantly lower, whereas CD8(+) T-cell levels, including activated CD38(+)CD8(+) T cell levels, were higher in the duodenum and jejunum, compared with the colon. After 6 months of cART, a significant but incomplete recovery of CD4(+) T cells was detected in the colon and peripheral blood but not in the duodenum. Failed restoration of the CD4(+) T-cell count in the duodenum was associated with nonspecific enteritis and CD8(+) T-cell activation.

Conclusions: Strategies that target inflammation and immune activation in the small intestine may be required to expedite CD4(+) T-cell recovery and improve therapeutic outcomes.
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http://dx.doi.org/10.1093/infdis/jit249DOI Listing
October 2013

Macrophage polarization at the crossroad between HIV-1 infection and cancer development.

Arterioscler Thromb Vasc Biol 2013 Jun;33(6):1145-52

AIDS Immunopathogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

Mononuclear phagocytes play a fundamental role in the tissue homeostasis and innate defenses against viruses and other microbial pathogens. In addition, they are likely involved in several steps of cancer development. Circulating monocytes and tissue macrophages are target cells of viral infections, including human cytomegalovirus, human herpes virus 8, and the HIV, and alterations of their functional and phenotypic properties are likely involved in many tissue-degenerative diseases, including atherosclerosis and cancer. Different tissue microenvironments as well as their pathological alterations can profoundly affect the polarization state of macrophages toward the extreme phenotypes conventionally termed M1 and M2. Thus, targeting disease-associated macrophages is considered a potential approach particularly in the context of cancer-associated tumor-associated macrophages, supporting malignant cell growth and progression toward a metastatic phenotype. Of note is the fact that tumor-associated macrophages isolated from established tumors display phenotypic and functional features similar to those of in vitro-derived M2-polarized cells. Concerning HIV-1 infection, viral eradication strategies in the context of combination antiretroviral therapy should also consider the possibility to deplete, at least transiently, certain mononuclear phagocytes subsets, although the possibility of distinguishing those that are either infected or pathogenically altered remains a goal of future research. In the present review, we will focus on the recent literature concerning the role of human macrophage polarization in viral infections and cancer.
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http://dx.doi.org/10.1161/ATVBAHA.112.300171DOI Listing
June 2013

M1 polarization of human monocyte-derived macrophages restricts pre and postintegration steps of HIV-1 replication.

AIDS 2013 Jul;27(12):1847-56

Objective: Functional polarization of human monocyte-derived macrophages (MDMs) into M1 cells leads to inhibition of R5 HIV-1 replication and viral DNA synthesis in comparison to control, unpolarized cells together with CD4 downregulation from the cell surface and upregulation of CCR5-binding chemokine secretion. We here investigated whether a postentry restriction of virus replication is also induced by M1 polarization of MDM.

Design: MDM were first polarized to M1 cells by 18 h stimulation with interferon-[gamma] and tumor necrosis factor-[alpha]; the cytokines were then removed and the cells were infected with vesicular stomatitis virus G-protein pseudotyped enhanced green fluorescence protein HIV-1 (HIV-GFP) generating a single-round infection cycle.

Methods: HIV-1 expression was monitored in terms of eGFP expression by fluorescence activated cell sorter (FACS) analysis and real-time PCR analysis of total HIV-1 gag DNA, 2-long terminal repeat DNA, proviral DNA, and multiply spliced RNA transcripts. Expression of apolipopoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), and APOBEC3A was tested by western blotting and FACS analysis.

Results: Inhibition of HIV-GFP expression was observed in M1-MDM along with impaired viral DNA synthesis, delayed proviral integration, and reduced proviral transcription. Although APOBEC3G levels were similar in M1 and unpolarized MDM, APOBEC 3A was selectively expressed only by M1 cells.

Conclusion: M1 polarization of in-vitro differentiated primary MDM determines a transient, but profound restriction of HIV-1 replication affecting multiple (entry and postentry) steps in the virus life cycle likely involving the upregulated expression of APOBEC3A.
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http://dx.doi.org/10.1097/QAD.0b013e328361d059DOI Listing
July 2013