Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.     

Role of Cell Surface Glycosaminoglycans of Human T Cells in Human Immunodeficiency Virus Type-1 (HIV-1) Infection

To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV-1 infection in human T cells, HIV-1 binding and infection were determined after treatment of T-cell lines and CD4 + T cells from normal peripheral blood mononuclear cells (PBMC) with GAG-degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV-1/IIIB to MT-4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage-tropic strain HIV-1/SF162 than that of the T-cell line-tropic strain HIV-1/IIIB. The binding of HIV-1/SF162 was about 100-fold less dependent on cell surface HS than HIV-1/IIIB. Human HTLV-I positive T-cell lines expressed more HS than HTLV-I negative T-cell lines or normal CD4 + T cells when stained with anti-HS mAbs against either native or heparitinase-treated HS. With the exception of endo-β-galactosidase (endo-β-gal), GAG-degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV-1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV-1 infection by facilitating HIV-1/IIIB binding to MT-4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV-1 binding inhibition, other GAG-like molecules may also be involved.     

Modeling of HIV-1 Infection: Insights to the Role of Monocytes/Macrophages,Latently Infected T4 Cells,and HAART Regimes

A novel dynamic model covering five types of cells and three connected compartments, peripheral blood (PB), lymph nodes (LNs), and the central nervous system (CNS), is here proposed. It is based on assessment of the biological principles underlying the interactions between the human immunodeficiency virus type I (HIV-1) and the human immune system. The simulated results of this model matched the three well-documented phases of HIV-1 infection very closely and successfully described the three stages of LN destruction that occur during HIV-1 infection. The model also showed that LNs are the major location of viral replication, creating a pool of latently infected T4 cells during the latency period. A detailed discussion of the role of monocytes/macrophages is made, and the results indicated that infected monocytes/macrophages could determine the progression of HIV-1 infection. The effects of typical highly active antiretroviral therapy (HAART) drugs on HIV-1 infection were analyzed and the results showed that efficiency of each drug but not the time of the treatment start contributed to the change of the turnover of the disease greatly. An incremental count of latently infected T4 cells was made under therapeutic simulation, and patients were found to fail to respond to HAART therapy in the presence of certain stimuli, such as opportunistic infections. In general, the dynamics of the model qualitatively matched clinical observations very closely, indicating that the model may have benefits in evaluating the efficacy of different drug therapy regimens and in the discovery of new monitoring markers and therapeutic schemes for the treatment of HIV-1 infection.     

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an “immune exhaustion”, with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28⁻CD57⁺CD8⁺ T cells between the groups. However, the frequency of Tim-3⁺CD8⁺ and Tim-3⁺CD4⁺ exhausted T cells, but not PD-1⁺ T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1⁺CD8⁺ T cells were directly associated with T cell immune activation in children. The frequency of Tim-3⁺CD8⁺ T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.     

T Cells Expressing Activated LFA-1 Are More Susceptible to Infection with Human Immunodeficiency Virus Type 1 Particles Bearing Host-Encoded ICAM-1

The incorporation of host-derived proteins in nascent human immunodeficiency virus type 1 (HIV-1) particles is a well-established phenomenon. We recently demonstrated that the physical presence of host-encoded ICAM-1 glycoproteins on HIV-1 leads to a significant increase in virus infectivity in an ICAM-1/LFA-1-dependent fashion (J.-F. Fortin, R. Cantin, G. Lamontagne, and M. Tremblay, J. Virol. 71:3588–3596, 1997). We show here that conversion of LFA-1 to high affinity for ICAM-1 with the use of anti-LFA-1 antibodies (clones NKI-L16 and MEM83) markedly enhances the susceptibility of different target T-lymphoid cell lines, as well as of primary peripheral blood mononuclear cells, to infection by ICAM-1-bearing HIV-1 particles (6- to 95-fold). It is known that T-cell receptor (TCR) cross-linking induces a transient increase in LFA-1 affinity for ICAM-1. Treatment of peripheral blood mononuclear cells with anti-TCR antibodies (clone OKT3) resulted in a transient increase in susceptibility to infection by ICAM-1-positive virions that parallels the previously reported kinetics of the LFA-1/ICAM-1 adhesion mechanism. Our results led us to postulate that the strong interaction taking place between virally incorporated ICAM-1 and cell surface-activated LFA-1 markedly enhances the efficiency of virus binding and entry, thus favoring greater infection by ICAM-1-bearing HIV-1 particles. In view of the knowledge that primary HIV-1 isolates harbor host-derived ICAM-1 on their surfaces, these results provide new information about the role of host-derived ICAM-1 in the life cycle of HIV-1 and how it could positively modulate the dynamics of the viral infection, mainly in cellular compartments, such as the lymphoid tissues, where the level of cellular activation is high and where the probability of encountering a T cell expressing the activated LFA-1 form is also elevated.In vivo, CD4⁺ T lymphocytes and monocytes-macrophages constitute the main reservoirs for the production and maintenance of human immunodeficiency virus type 1 (HIV-1) (48, 54). Infection of these cells occurs following the high-affinity interaction between the viral surface gp120 and the cell surface CD4 molecule (15). However, in order for the fusion to occur, the sole interaction between gp120 and CD4 is not sufficient (40), and the involvement of other molecules is required. These other cellular components, the so-called coreceptors, have been recently identified and characterized. Formerly called LESTR/HUMSTR/fusin, the chemokine receptor CXCR4 has been shown to act as the coreceptor for T-cell-tropic strains of HIV-1 (22). For macrophage-tropic HIV-1 isolates, the CCR5 molecule has been identified as the major coreceptor (16, 19), even though CCR3 and CCR2b are also used, but to a lesser extent (14, 18). Following ligation of gp120 with CD4, a high-affinity binding site for the chemokine receptor is created, thus leading to membrane fusion and virus entry (36, 58, 59). Besides these essential elements for viral entry, other cellular molecules could play important, although accessory, roles during the process of virus uptake.It has been known for a while that HIV-1 can incorporate, besides its surface glycoproteins, a vast array of cell membrane molecules while budding out from the infected cell. For example, major histocompatibility complex class II (MHC-II) DR molecules were the first host constituents found embedded within HIV-1 particles and these were identified as a potential source of false-positive reactions in enzymatic screening tests (31). Many other cellular structures were found to be acquired by newly formed HIV-1, such as HLA-DP and -DQ, β₂-microglobulin, CD44, CD55, and CD59, as well as LFA-1 and ICAM-1 adhesion molecules (6, 11, 12, 21, 29, 33, 52). It has also been suggested that the profile of virion-bound cellular constituents could be used as a marker to identify the virus-producing cell (1).Recently, several studies investigated the functionality of host-derived molecules when present on the virion surface. The first, although indirect, evidence of the functionality of virally incorporated adhesion molecules came from the demonstration that anti-LFA-1 antibodies can act synergistically with antiserum to neutralize HIV-1 particles (28). More direct proof was provided by the demonstration that an increase in virion-incorporated HLA-DR and ICAM-1 resulted in enhanced infectivity toward CD4-negative cell lines (12). Saiffudin et al. demonstrated that CD55 and CD59, two glycosylphosphatidylinositol-linked complement control proteins, can protect HIV-1 from complement-mediated virolysis when incorporated into budding virions (52), while virion-incorporated host MHC-II molecules were shown to present bacterial superantigens (50). We have been able to demonstrate, by using mutagenized cell lines, that incorporation of MHC-II molecules within the viral envelope enhances the process of viral infection (9). Recently, we developed a transient-transfection-and-expression system that permits the production of virions differing only by the absence or the presence of a specific cell surface molecule on their surfaces. By using this new technical approach, we found that acquisition of cellular HLA-DR1 molecules by budding HIV-1 is associated with a 1.6- to 2.5-fold increase in virus infectivity (10). Moreover, we have shown that incorporation of host-derived ICAM-1 by progeny viruses leads to a 5- to 10-fold increase in HIV-1 infectivity, caused by an interaction between virally incorporated ICAM-1 and cell surface LFA-1 (23), an observation which has been corroborated by another group (49). This finding has great clinical relevance, considering that ICAM-1 is acquired by clinical HIV-1 isolates grown on primary mononuclear cells (4, 11, 24) and the in vivo HIV-1-producing cells are activated CD4⁺ T cells and macrophages, cells which are both known to express high levels of ICAM-1 (55). Therefore, it is likely that HIV-1 isolates found in vivo carry on their surfaces host-derived ICAM-1 glycoproteins.The counterreceptor for ICAM-1 is LFA-1, a member of the integrin family that is expressed mainly on lymphocytes, granulocytes, monocytes, and macrophages, with elevated levels on memory T cells (53). The activation of leukocytes with various agents like phorbol esters and chemoattractant, or cross-linking of specific surface receptors such as the T-cell receptor (TCR)/CD3 complex, CD2, and MHC-II, induces LFA-1-mediated binding to ICAM-1 (17). This transient change in ICAM-1 binding is thought to involve both a variation in the affinity of LFA-1 for ICAM-1 caused by a conformational change and an increase in avidity mediated by clustering of the molecules (20). This dynamic regulation of integrins allows the cells that bear these molecules to convert rapidly from a nonadherent to an adherent phenotype and vice versa. Since LFA-1 can be expressed in two different conformational states, i.e., low versus high affinity for ICAM-1, we therefore examined whether the activation state of LFA-1 on the target cell surface could affect the overall susceptibility to infection by ICAM-1-bearing HIV-1 particles.     

In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses,Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

Background

The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission.

Methodology

The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited.

Findings

Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4⁺ and CD8⁺ T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4⁺ T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4⁺ T cell depletion which renders the newly activated HIV-specific CD4⁺ T cells prime targets for elimination.

Conclusion

Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.     

Influenza-Infected Neutrophils within the Infected Lungs Act as Antigen Presenting Cells for Anti-Viral CD8+ T Cells

Influenza A virus (IAV) is a leading cause of respiratory tract disease worldwide. Anti-viral CD8⁺ T lymphocytes responding to IAV infection are believed to eliminate virally infected cells by direct cytolysis but may also contribute to pulmonary inflammation and tissue damage via the release of pro-inflammatory mediators following recognition of viral antigen displaying cells. We have previously demonstrated that IAV antigen expressing inflammatory cells of hematopoietic origin within the infected lung interstitium serve as antigen presenting cells (APC) for infiltrating effector CD8⁺ T lymphocytes; however, the spectrum of inflammatory cell types capable of serving as APC was not determined. Here, we demonstrate that viral antigen displaying neutrophils infiltrating the IAV infected lungs are an important cell type capable of acting as APC for effector CD8⁺ T lymphocytes in the infected lungs and that neutrophils expressing viral antigen as a result of direct infection by IAV exhibit the most potent APC activity. Our findings suggest that in addition to their suggested role in induction of the innate immune responses to IAV, virus clearance, and the development of pulmonary injury, neutrophils can serve as APCs to anti-viral effector CD8⁺ T cells within the infected lung interstitium.     

Expression of Target Antigen for Epstein-Barr Virus-Specific Cytotoxic T Cells on BJAB Cells Freshly Infected with EBV

The target antigen for Epstein-Barr virus (EBV)-specific cytotoxic T cells (Tc) was expressed on BJAB cells exposed to the B95-8 strain of EBV for at least one hour. Ultraviolet-light (UV)-irradiated noninfectious B95-8 virus also induced the target antigen on BJAB cells. Cold target competition tests suggested that the target antigen expressed on EBV-infected BJAB cells was distinct from the lymphocyte-detected membrane antigen (LYDMA) which was also recognized by the EBV-specific Tc and expressed on autologous EBV-transformed lymphoblastoid cell line (LCL) cells. Neither of these target antigens for EBV-specific Tc was detected on the surface of EBV-genome positive BJAB cells which had been kept in a long-term culture after EBV-infection. Thus, the virion antigen, especially the EBV-membrane antigen (MA), is a possible candidate for the target antigen expressed on EBV-infected BJAB cells. Lysis of EBV-infected BJAB cells was inhibited by target cell treatment with anti-β2 microglobulin (anti-β2M) antibody and induction of the effector Tc was dependent on the donor individual. These resalts suggested the possibility that the Tc recognizing EBV-infected BJAB cells are restricted by the major histocompatibility complex (MHC).     

Influenza A Virus Infection of Human Primary Dendritic Cells Impairs Their Ability to Cross-Present Antigen to CD8 T Cells

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection.     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

Non-Transferrin-Bound Iron (NTBI) Uptake by T Lymphocytes: Evidence for the Selective Acquisition of Oligomeric Ferric Citrate Species

Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI). NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.     

Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells, using a virion-free pathway. Here, we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles or infectious RNA which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells was mediated by a contact-dependent RNA transfer to pDC, which was sensitive to a TLR7 inhibitor. This was inhibited by drugs affecting the cytoskeleton and membrane cholesterol. We further demonstrate that a unique viral protein with ribonuclease activity, the viral E^rns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. We propose that this pathway of activation could be of particular importance for viruses which tend to be mostly cell-associated, cause persistent infection, and are non-cytopathogenic.     

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4⁺ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4⁺ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-α/β cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.     

Clearance of Measles Virus from Persistently Infected Cells by Short Hairpin RNA

Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are “cured.” This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA.The neurodegenerative human disease subacute sclerosing panencephalitis (SSPE) occurs with an incidence rate of approximately 1:10,000 after infection with wild-type measles virus (MV) (4, 38). The course of the illness is quite variable, usually lasting from 1 to 3 years. Much more rapid forms that lead to death within a few months as well as prolonged courses with a duration of more than 20 years have been described (40). Neuropathological findings include diffuse encephalitis, affecting both the gray and white matters, characterized by perivascular cuffing and diffuse lymphocytic infiltrations. Neurons, oligodendrocytes, fibrous astrocytes, and some brain microvascular endothelial cells contain large aggregates of intranuclear inclusion bodies consisting of MV nucleocapsid structures (1, 16). In these persistently infected cells, viral ribonucleoprotein particles (RNPs) replicate intracellularly, whereas the budding of complete viruses and cell-cell fusion are not observed. A characteristic feature of this central nervous system disease is that the expression of viral envelope proteins (matrix [M], fusion [F], and hemagglutinin [H] proteins) is restricted by various means. In particular, the M protein and the cytoplasmic part of the F protein harbor single or hypermutations or deletions, which prevent their proper expression (2, 3, 9, 10). The lack of M reduces budding, supports cell fusion, and enhances the intracellular replication of RNPs (7, 8, 32, 37). As far as is known, the cell-to-cell spread of infectivity in the human brain occurs in the presence of normal cellular and strong humoral antiviral immune responses with very high anti-MV antibody titers in the cerebrospinal fluid. This, however, cannot prevent virus spread.A variety of approaches to the treatment of SSPE have been attempted, but an evaluation of their efficiency has been extremely difficult, since clinical trials are based on small numbers of patients, the course of SSPE is highly variable, and spontaneous remissions may also occur. Intrathecal or intraventricular administration of alpha interferon, inosiplex, and/or ribavirin is a common regimen, but despite many efforts, the establishment of an effective therapy has not been possible. Since the immune systems of the patients appear normal, and given the fact that virus spreads in the form of intracellular RNPs, a promising specific therapy must target this intracellular replication of MV.RNA interference (RNAi) may provide such a means and has already been used successfully to inhibit the expression of a number of viral infections, including the Ebola, influenza A, hepatitis B and C, human immunodeficiency, respiratory syncytial, and West Nile viruses, and several RNAi-based therapeutics are already in preclinical test phases (for reviews, see references 6 and 24). Small interfering RNAs (siRNAs) have also been described to be active against MV (20, 29, 32), including an MV isolate from an SSPE patient (SSPE-Kobe-1) (28). In the latter approach, the authors generated recombinant adenoviruses (rAdV) expressing siRNA against MV L mRNA and assessed them in freshly infected Vero/SLAM cells. In contrast to this work, we constructed lentiviral vectors expressing short hairpin RNAs (shRNAs) and transduced persistently infected human NT2 cells with these vectors. This lentiviral approach provided the proof of principle that a preexisting persistent MV infection can be cured by shRNA.     

Abortive Infection of F-Plasmid-Containing Escherichia coli Cells by Bacterial Virus T7 Is Determined by the Right End of T7 Gene 1

Phage T7 infects male (F-plasmid-carrying) Escherichia coli cells abortively, whereas the closely related phage T3 grows normally. The inability or ability of phage to replicate in male host cells depends on whether the right end of gene 1 (coding for the phage-specific RNA polymerase) consists of T7 or T3 DNA base sequences.