Influence of fibronectin on HIV-1 infection and capability of binding to platelets

We have previously demonstrated that fibronectin (FN) can bind HIV-1 envelope proteins, in particular gp120. The aim of the present study was to determine some biological effects of this phenomenon. Pretreating HIV-1 with human FN increased the infectivity of HIV-1, when a low concentration of the virus was used. In contrast, an RGD-containing pentapeptide (Gly-Arg-Gly-Asp-Ser), which is a fundamental binding site of FN, reduced the infectivity of a suspension of HIV-1 at high concentrations of the virus. It is likely that FN bridges the cell surface and the virions, while the RGD-containing pentapeptide may saturate the HIV-1 binding sites for cell surface receptors. Moreover, gp120 was bound to the FN present on the surface of platelets. The specifity of this binding was confirmed by the inhibition obtained by pretreating platelets with anti-FN antibodies. The consequence of the surface modifications of the platelets could explain the thrombocytopenia that frequently occurs in patients infected with HIV and suggests also the possibility that platelets could be a vehicle for the virus in the circulation.     

Expression of fibronectin and its receptor in some tumour cell lines and in HIV-1-infected cells

The aim of our study was to evaluate the levels of fibronectin (FN) and its classic receptor (FNR) in various transformed cells lines, especially of leukemic origin, and also the influence of HIV-1 replication on the expression of these proteins (in particular on H9-V cells, chronically infected with HIV-1, and acutely infected MT-4 cells). Monoclonal antibodies were used for indirect immunofluorescence tests; the fluorescein-conjugated recombinant p14, the product of the HIV gene tat, was used as a molecular probe. The results demonstrated a high variability of FN and FNR expression among the various cellular lines studied. Moreover, deficits of such adhesive proteins did not necessarily correlate with a severe reduction of the corresponding receptor. HIV-1 replication in MT-4 and H9-V cells increased the expression of FNR. This seems to correlate with p14-induced phenomena because pretreatment of H9-V cells with recombinant p14 showed an enhancing effect on the expression of this receptor.     

HCV infective virions can be carried by human platelets

It has been previously demonstrated that platelets (PLTs) can bind and transport HIV-1 infectious virions. Hepatitis C virus (HCV)-HIV-1 co-infection occurs frequently among users of illicit intravenous drugs, thereby increasing the severity of HIV disease and the evolution towards chronic active hepatitis and hepatocellular carcinoma of HCV-related hepatitis. In the present study we investigated whether or not PLTs can carry HCV, and studied the binding mechanisms. Purified PLTs, obtained from healthy donors, HCV negative and HIV negative, were adsorbed with HCV-containing serum and then employed to infect a THP-1 monocytoid cell line. Replication of HCV was observed as shown by positivity for the E2 antigen within THP-1 cells, by indirect immunofluorescence; moreover, HCV-RNA was detected in supernatants of THP-1 cells at day 7 post-incubation with HCV-adsorbed PLTs. The binding of HCV to PLTs seems to involve fibronectin (FN), as already shown in the case of HIV-1. Indeed, treatment with RGD (Gly-Arg-Gly-Asp-Ser), the key oligopeptide of FN binding, inhibits the ability of HCV to be carried by PLTs in infective forms; the same phenomenon occurs with Mabs to FN. Moreover the infection of THP-1 cells seems to increase FN surface expression, as demonstrated by immunofluorescence tests.     

HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.     

Enhancement of Human Immunodeficiency Virus Type 1 (HIV-1) Infection via Increased Membrane Fluidity by a Cationic Polymer

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV-1 infection. Although virus-cell binding was significantly inhibited by PEI, HIV-1 infection in human T-cell lines such as MT-4 and MOLT-4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV-1 infection was examined using HIV-1 chronically infected cells (MOLT-4/HIV-1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT-4/HIV-1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV-1 to CD4-positive cells by PEI was due to the shedding of gp120 from HIV-1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV-1 infection. Similarly, dissociation of gp120 from MOLT-4/HIV-1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV-1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.     

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.     

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.     

Enigma of HIV-1 latent infection in astrocytes: an in-vitro study using protein kinase C agonist as a latency reversing agent

Purging HIV-1 to cure the infection in patients undergoing suppressive antiretroviral therapy requires targeting all possible viral reservoirs. Other than the memory CD4⁺ T cells, several other HIV-1 reservoirs have been identified. HIV-1 infection in the brain as a reservoir is well documented, but not fully characterized. There, microglia, perivascular macrophages, and astrocytes can be infected by HIV-1. HIV-1 infection in astrocytes has been described as a nonproductive and primarily a latent infection. Using primary human astrocytes, we investigated latent HIV-1 infection and tested phorbol 12-myristate 13-acetate (PMA), a protein kinase C agonist, as an HIV-1-latency- reversing agent in infected astrocytes. Chloroquine (CQ) was used to facilitate initial HIV-1 escape from endosomes in astrocytes. CQ significantly increased HIV-1 infection. But treatment with PMA or viral Tat protein was similar to untreated HIV-1-infected astrocytes. Long-term follow-up of VSV-envelope-pseudotyped HIV-1 infected astrocytes showed persistent infection for 110 days, indicating the active state of the virus.     

Live-cell real-time imaging reveals role of mitochondria in cell-to-cell transmission of HIV-1

We used live-cell, real-time fluorescence imaging of co-cultures of HIV-1 infected T cells and uninfected target cells to examine the action of mitochondria during cell-to-cell transmission of the virus. We find that mitochondria of HIV infected cells enter uninfected target cells and advance viral spread. We show that human mitochondria serve as viral reservoirs and carriers and that they can move between cells. This was confirmed by our results that purified mitochondria from HIV infected cells are infectious, and that mitochondrial inhibitors block HIV transmission. Viral infection and replication in the target cells were verified by syncytial formation and HIV-1 core protein p24 production. Our results offer new insights into the cellular mechanisms of viral transmission and identify mitochondria as new host targets for viral infection.     

Suppression of HIV-1 Integration by Targeting HIV-1 Integrase for Degradation with A Chimeric Ubiquitin Ligase

Human immunodeficiency virus(HIV) attacks human immune system and causes life-threatening acquired immune deficiency syndrome(AIDS). Treatment with combination antiretroviral therapy(cART) could inhibit virus growth and slow progression of the disease, however, at the same time posing various adverse effects. Host ubiquitin-proteasome pathway(UPP) plays important roles in host immunity against pathogens including viruses by inducing degradation of viral proteins. Previously a series of methods for retargeting substrates for ubiquitin-proteasome degradation have been successfully established. In this study, we attempted to design and construct artificial chimeric ubiquitin ligases(E3 s) based on known human E3 s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation.Herein, a series of prototypical chimeric E3 s have been designed and constructed, and original substrate-binding domains of these E3 s were replaced with host protein domains which interacted with viral proteins. After functional assessment screening, 146 LI was identified as a functional chimeric E3 for HIV-1 NL4-3 integrase. 146 LI was then further optimized to generate 146 LIS(146 LI short) which has been shown to induce Lys48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells. Lymphocyte cells with 146 LIS knock-in generated by CRISPR/Cas-mediated homology-directed repair(HDR) showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity. Our study successfully obtained an artificial chimeric E3 which can induce Lys48-specific polyubiquitination and proteasome-mediated degradation of HIV-1 NL4-3 integrase, thus effectively inhibiting viral DNA integration and viral replication upon virus infection.