Production of lentiviral vectors with enhanced efficiency to target dendritic cells by attenuating mannosidase activity of mammalian cells

Background  

Dendritic cells (DCs) are antigen-presenting immune cells that interact with T cells and have been widely studied for vaccine applications. To achieve this, DCs can be manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired antigen-specific T cell response, which gives this approach great potential to fight diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs through a specific interaction with DC-SIGN, a surface molecule predominantly expressed by DCs. We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that an increase in high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit mannosidase, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production.     

Measles virus glycoprotein-pseudotyped lentiviral vectors are highly superior to vesicular stomatitis virus G pseudotypes for genetic modification of monocyte-derived dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells capable of promoting or regulating innate and adaptive immune responses against non-self antigens. To better understand the DC biology or to use them for immune intervention, a tremendous effort has been made to improve gene transfer in these cells. Lentiviral vectors (LVs) have conferred a huge advantage in that they can transduce nondividing cells such as human monocyte-derived DCs (MDDCs) but required high amounts of viral particles and/or accessory proteins such as Vpx or Vpr to achieve sufficient transduction rates. As a consequence, these LVs have been shown to cause dramatic functional modifications, such as the activation or maturation of transduced MDDCs. Taking advantage of new pseudotyped LVs, i.e., with envelope glycoproteins from the measles virus (MV), we demonstrate that MDDCs are transduced very efficiently with these new LVs compared to the classically used vesicular stomatitis virus G-pseudotyped LVs and thus allowed to achieve high transduction rates at relatively low multiplicities of infection. Moreover, in this experimental setting, no activation or maturation markers were upregulated, while MV-LV-transduced cells remained able to mature after an appropriate Toll-like receptor stimulation. We then demonstrate that our MV-pseudotyped LVs use DC-SIGN, CD46, and CD150/SLAM as receptors to transduce MDDCs. Altogether, our results show that MV-pseudotyped LVs provide the most accurate and simple viral method for efficiently transferring genes into MDDCs without affecting their activation and/or maturation status.     

Heterologous human immunodeficiency virus type 1 lentiviral vectors packaging a simian immunodeficiency virus-derived genome display a specific postentry transduction defect in dendritic cells

Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV(MAC251)), we asked whether heterologous virion particles in which trans-acting factors belonged to HIV-1 and cis elements belonged to SIV(MAC251) (HIV-siv) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.     

Maturation of blood-derived dendritic cells enhances human immunodeficiency virus type 1 capture and transmission

Dendritic cells (DCs) are specialized antigen-presenting cells. However, DCs exposed to human immunodeficiency virus type 1 (HIV-1) are also able to transmit a vigorous cytopathic infection to CD4(+) T cells, a process that has been frequently related to the ability of DC-SIGN to bind HIV-1 envelope glycoproteins. The maturation of DCs can increase the efficiency of HIV-1 transmission through trans infection. We aimed to comparatively study the effect of maturation in monocyte-derived DCs (MDDCs) and blood-derived myeloid DCs during the HIV-1 capture process. In vitro capture and transmission of envelope-pseudotyped HIV-1 and its homologous replication-competent virus to susceptible target cells were assessed by p24(gag) detection, luciferase activity, and both confocal and electron microscopy. Maturation of MDDCs or myeloid DCs enhanced the active capture of HIV-1 in a DC-SIGN- and viral envelope glycoprotein-independent manner, increasing the life span of trapped virus. Moreover, higher viral transmission of mature DCs to CD4(+) T cells was highly dependent on active viral capture, a process mediated through cholesterol-enriched domains. Mature DCs concentrated captured virus in a single large vesicle staining for CD81 and CD63 tetraspanins, while immature DCs lacked these structures, suggesting different intracellular trafficking processes. These observations help to explain the greater ability of mature DCs to transfer HIV-1 to T lymphocytes, a process that can potentially contribute to the viral dissemination at lymph nodes in vivo, where viral replication takes place and there is a continuous interaction between susceptible T cells and mature DCs.     

Construction of stable producer cells to make high-titer lentiviral vectors for dendritic cell-based vaccination

Lentiviral vectors (LVs) enveloped with an engineered Sindbis virus glycoprotein can specifically bind to dendritic cells (DCs) through the surface receptor DC-SIGN and induce antigen expression, thus providing an efficient method for delivering DC-directed vaccines. In this study, we constructed a stable producer line (LV-MGFP) for synthesizing DC-SIGN-targeted HIV-1-based LVs (DC-LVs) encoding green fluorescent protein (GFP) by a concatemeric array transfection technique. We demonstrated that the established stable clones could routinely produce vector supernatants with titers above 10(7) transduction units per milliliter (TU/mL) during a continuous 3-month cell passage. The producer cells were also capable of generating similar titers of DC-LVs in serum-free medium. Moreover, the addition of 1-deoxymannojirimycin (DMJ) enabled the producer cells to manufacture DC-LVs with both improved titers and enhanced potency to evoke antigen-specific CD8(+) T cell responses in mice. The stable lines could accommodate the replacement of the internal murine stem cell virus (MSCV) promoter with the human ubiquitin-C (Ubi) promoter in the lentiviral backbone. The resulting DC-LVs bearing Ubi exhibited the enhanced potency to elicit vaccine-specific immunity. Based on accumulated evidence, our studies support the application of this production method in manufacturing DC-LVs for preclinical and clinical testing of novel DC-based immunization.     

Processing and presentation of exogenous HLA class I peptides by dendritic cells from human immunodeficiency virus type 1-infected persons

Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A*0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8(+) T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV-1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8(+) T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection.     

A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus into Dendritic Cells

Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation.     

Exploiting dendritic cells for cancer immunotherapy: genetic modification of dendritic cells

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct characteristics and possible advantages and disadvantages in respect to their use in DC-based immunotherapy.     

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-SIGN internalization signals

Dengue virus (DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pH-dependent fusion into DCs.     

Expression of the self-marker CD47 on dendritic cells governs their trafficking to secondary lymphoid organs

Dendritic cells (DCs) capture and process Ag in the periphery. Thus, traffic through lymphatic vessels is mandatory before DCs relocate to lymph nodes where they are dedicated to T-cell priming. Here, we show that the ubiquitous self-marker CD47 selectively regulates DC, but not T and B cell trafficking across lymphatic vessels and endothelial barriers in vivo. We find an altered skin DC migration and impaired T-cell priming in CD47-deficient mice at steady state and under inflammatory conditions. Competitive DC migration assays and active immunization with myeloid DCs demonstrate that CD47 expression is required on DCs but not on the endothelium for efficient DC trafficking and T-cell responses. This migratory defect correlates with the quasi-disappearance of splenic marginal zone DCs in nonmanipulated CD47-deficient mice. Nonetheless, CCR7 expression and CCL19-driven chemotaxis remain intact. Our data reveal that CD47 on DCs is a critical factor in controlling migration and efficient initiation of the immune response.     

A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1-derived lentiviral vectors

Lentiviral vectors derived from the human immunodeficiency type 1 virus (HIV-1 LV) are among the finest tools available today for the genetic modification of human monocyte-derived dendritic cells (MDDCs). However, this process is largely inefficient because MDDCs show a strong resistance to HIV-1 transduction. Here we describe a step-by-step protocol from the production of LVs to cell transduction that allows the efficient genetic modification of MDDCs. This protocol can be completed in 23 d from the initial phase of LV production to the final analysis of the results of MDDC transduction. The method relies on the simultaneous addition of HIV-1 LVs along with noninfectious virion-like particles carrying Vpx, a nonstructural protein encoded by the simian immunodeficiency virus (Vpx-VLPs). When thus provided in target cells, Vpx exerts a strong positive effect on incoming LVs by counteracting the restriction present in MDDCs; accordingly, 100% of cells can be transduced with low viral inputs. Vpx-VLPs will improve the efficiency of LV-mediated transduction of MDDCs with vectors for both ectopic gene expression and depletion studies.     

Virus infection of dendritic cells: portal for host invasion and host defense

Dendritic cells (DCs) act as a portal for virus invasion and as the most potent antigen-presenting cells in antiviral host defense. Human immunodeficiency virus (HIV)-1 has served as the paradigm for virus interaction with DCs. HIV-1 infection of DCs via its primary CD4 receptor and secondary chemokine receptors leads to full virus replication (cis infection), whereas binding to C-type lectin receptors results both in cis replication, as well as transfer and replication of virus in CD4(pos) T cells (trans infection). DCs respond to this invasion by processing viral proteins through MHC class I and II pathways and undergoing a maturation that enhances their presentation of antigen to T cells for induction of adaptive antiviral immunity. HIV-1 and other viruses have evolved mechanisms to subvert this immune function. Engineering of DCs with various forms of viral immunogens and co-treatment with cytokines and chemokines is being used as an immunotherapy for HIV-1 and other viral infections.     

Dendritic-cell interactions with HIV: infection and viral dissemination

Dendritic cells (DCs) are crucial for the generation and the regulation of adaptive immunity. Because DCs have a pivotal role in marshalling immune responses, HIV has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Defining the mechanisms that underlie cell-cell transmission of HIV and understanding the role of DCs in this process should help us in the fight against HIV infection. This Review highlights the latest advances in our understanding of the interactions between DCs and HIV, focusing on the mechanisms of DC-mediated viral dissemination.     

Infection of dendritic cells (DCs), not DC-SIGN-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to T cells

The C-type lectin DC-SIGN expressed on immature dendritic cells (DCs) captures human immunodeficiency virus (HIV) particles and enhances the infection of CD4+ T cells. This process, known as trans-enhancement of T-cell infection, has been related to HIV endocytosis. It has been proposed that DC-SIGN targets HIV to a nondegradative compartment within DCs and DC-SIGN-expressing cells, allowing incoming virus to persist for several days before infecting target cells. In this study, we provide several lines of evidence suggesting that intracellular storage of intact virions does not contribute to HIV transmission. We show that endocytosis-defective DC-SIGN molecules enhance T-cell infection as efficiently as their wild-type counterparts, indicating that DC-SIGN-mediated HIV internalization is dispensable for trans-enhancement. Furthermore, using immature DCs that are genetically resistant to infection, we demonstrate that several days after viral uptake, HIV transfer from DCs to T cells requires viral fusion and occurs exclusively through DC infection and transmission of newly synthesized viral particles. Importantly, our results suggest that DC-SIGN participates in this process by cooperating with the HIV entry receptors to facilitate cis-infection of immature DCs and subsequent viral transfer to T cells. We suggest that such a mechanism, rather than intracellular storage of incoming virus, accounts for the long-term transfer of HIV to CD4+ T cells and may contribute to the spread of infection by DCs.     

Human immunodeficiency virus type 1 replication in dendritic cell-T-cell cocultures is increased upon incorporation of host LFA-1 due to higher levels of virus production in immature dendritic cells

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.     

Functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells

Dendritic cells (DCs) potently stimulate the transmission of human immunodeficiency virus type 1 (HIV-1) to CD4(+) T cells. Immature DCs (iDCs) located in submucosal tissues can capture HIV-1 and migrate to lymphoid tissues, where they become mature DCs (mDCs) for effective antigen presentation. DC maturation promotes HIV-1 transmission; however, the underlying mechanisms remain unclear. Here we have compared monocyte-derived iDCs and mDCs for their efficiencies and mechanisms of HIV-1 transmission. We have found that mDCs significantly facilitate HIV-1 endocytosis and efficiently concentrate HIV-1 at virological synapses, which contributes to mDC-enhanced viral transmission, at least in part. mDCs were more efficient than iDCs in transferring HIV-1 to various types of target cells independently of C-type lectins, which partially accounted for iDC-mediated HIV-1 transmission. Efficient HIV-1 trans-infection mediated by iDCs and mDCs required contact between DCs and target cells. Moreover, rapid HIV-1 degradation occurred in both iDCs and mDCs, which correlated with the lack of HIV-1 retention-mediated long-term viral transmission. Our results provide new insights into the mechanisms underlying DC-mediated HIV-1 transmission, suggesting that HIV-1 exploits mDCs to facilitate its dissemination within lymphoid tissues.     

MHC class I endosomal and lysosomal trafficking coincides with exogenous antigen loading in dendritic cells

Background

Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined.

Methodology/Principal Findings

In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing.

Conclusions/Significance

We conclude that DCs have ‘hijacked’ and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place.     

Role of dendritic cells in the induction of regulatory T cells

Dendritic cells (DCs) play a key role in initiating immune responses and maintaining immune tolerance. In addition to playing a role in thymic selection, DCs play an active role in tolerance under steady state conditions through several mechanisms which are dependent on IL-10, TGF-β, retinoic acid, indoleamine-2,3,-dioxygenase along with vitamin D. Several of these mechanisms are employed by DCs in induction of regulatory T cells which are comprised of Tr1 regulatory T cells, natural and inducible foxp3+ regulatory T cells, Th3 regulatory T cells and double negative regulatory T cells. It appears that certain DC subsets are highly specialized in inducing regulatory T cell differentiation and in some tissues the local microenvironment plays a role in driving DCs towards a tolerogenic response. In this review we discuss the recent advances in our understanding of the mechanisms underlying DC driven regulatory T cell induction.     

Differences and similarities in viral life cycle progression and host cell physiology after infection of human dendritic cells with modified vaccinia virus Ankara and vaccinia virus

Modified vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus (VV) that has attracted significant attention as a candidate viral vector vaccine for immunization against infectious diseases and treatment of malignancies. Although MVA is unable to replicate in most nonavian cells, vaccination with MVA elicits immune responses that approximate those seen after the administration of replication-competent strains of VV. However, the mechanisms by which these viruses elicit immune responses and the determinants of their relative immunogenicity are incompletely understood. Studying the interactions of VV and MVA with cells of the human immune system may elucidate these mechanisms, as well as provide a rational basis for the further enhancement of the immunogenicity of recombinant MVA vectors. Toward this end, we investigated the consequences of MVA or VV infection of human dendritic cells (DCs), key professional antigen-presenting cells essential for the generation of immune responses. We determined that a block to the formation of intracellular viral replication centers results in abortive infection of DCs with both VV and MVA. MVA inhibited cellular protein synthesis more rapidly than VV and displayed a distinct pattern of viral protein expression in infected DCs. MVA also induced apoptosis in DCs more rapidly than VV, and DC apoptosis after MVA infection was associated with an accelerated decline in the levels of intracellular Bcl-2 and Bcl-X(L). These findings suggest that antigen presentation pathways may contribute differentially to the immunogenicity of VV and MVA and that targeted modifications of virus-induced DC apoptosis may further increase the immunogenicity of MVA-vectored vaccines.     

Adeno-associated virus type 2-mediated gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts

Adeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, nonpathogenic human parvovirus that is currently in use as a vector for human gene therapy. However, the transduction efficiency of AAV vectors in different cell and tissue types varies widely. In addition to the lack of expression of the viral receptor and coreceptors and the rate-limiting viral second-strand DNA synthesis, which have been identified as obstacles to AAV-mediated transduction, we have recently demonstrated that impaired intracellular trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J. Hansen, K. Qing, H. J. Kwon, C. Mah, and A. Srivastava, J. Virol. 74:992-996, 2000). In this report, we document that escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293. While virions were found in both early and late endosomes or lysosomes of infected 293 cells, they were localized predominantly to the early endosomes in NIH 3T3 cells. Moreover, treatment of cells with bafilomycin A1 (Baf), an inhibitor of the vacuolar H(+)-ATPase and therefore of endosomal-lysosomal acidification, decreased the transduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated transduction in general, virions were detected in late endosomes and lysosomes, and these cells became sensitive to Baf-mediated inhibition of transduction. Thus, HU treatment overcomes defective endocytic processing of AAV in murine fibroblasts. These studies provide insights into the underlying mechanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy.