Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.     

Development of an avian leukosis-sarcoma virus subgroup A pseudotyped lentiviral vector

We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 x 10(3) infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1 pol gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 10(5) IU/ml and can be concentrated by ultracentrifugation to titers of 10(7) IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the beta-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.     

Inactivation of viruses in red cell and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonates.

Aluminum phthalocyanine tetrasulfonates (AIPcS) are photoactive compounds with absorption maxima at 665-675 nm. The inactivation of viruses (vesicular stomatitis virus, VSV; human immunodeficiency virus, HIV) added to either whole blood or red blood cell concentrates (RBCC) and platelet concentrates (PC) on treatment with tetrasulfonated AIPc (AIPcS4) was evaluated. Treatment of RBCC with 10 microM AIPcS4 and 44 J/cm2 visible light resulted in the inactivation of greater than or equal to 10(5.5) infectious doses (TCID50) of cell-free VSV, greater than or equal to 10(5.6) TCID50 of cell-associated VSV, and greater than or equal to 10(4.7) TCID50 of cell-free sindbis virus. Both greater than or equal to 10(4.2) TCID50 of cell-free and greater than or equal to 10(3.6) TCID50 of cell-associated forms of HIV were also shown to be inactivated. Encephalomyocarditis virus, used as a model for nonenveloped viruses, was not inactivated. Equivalent virus kill with Photofrin II required a substantially higher concentration of dye and longer exposure to visible light. Following AIPcS4 treatment, red cell integrity was well maintained as judged by the low level (less than 2%) of hemoglobin release immediately following treatment and on subsequent storage, by measurements of erythrocyte osmotic fragility, and by the normal recovery and circulatory survival on infusion of treated, autologous red blood cells in baboons. Treatment of PC with 10 microM AIPcS4 and 44 J/cm2 visible light also resulted in effective virus kill (greater than or equal to 10(5.5) TCID50) of VSV; however, both the rate and extent of platelet aggregation in response to collagen addition declined by at least 50%. Based on these results, further characterization of AIPcS4-treated RBCC is justified.     

Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells

Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression.     

Characterization of envelope glycoprotein mutants for human T-cell leukemia virus type 1 infectivity and immortalization

The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4(+) lymphocytes. These results indicate that HTLV-1 spread is required for immortalization.     

Mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus TRIM5alpha

Rhesus TRIM5α (rhTRIM5α), but not human TRIM5α (huTRIM5α), potently inhibits human immunodeficiency virus (HIV) infection and is thus a potentially valuable therapeutic tool. Primary human CD4 T cells engineered to express rhTRIM5α were highly resistant to cell-free HIV type 1 (HIV-1) infection. However, when cocultured with unmodified T cells, rhTRIM5α-expressing cells became highly permissive to HIV-1 infection. Physical separation of rhTRIM5α-expressing cells and unmodified cells revealed that rhTRIM5α efficiently restricts cell-free but not cell-associated HIV transmission. Furthermore, we observed that HIV-infected human cells could infect rhesus CD4 T cells by cell-to-cell contact, but the infection was self-limiting. Subsequently, we noted that a spreading infection ensued when HIV-1-infected rhTRIM5α-expressing human cells were cultured with huTRIM5α- but not rhTRIM5α-expressing cells. Our results suggest that cell-associated HIV transmission in humans is blocked only when both donor and recipient cells express rhTRIM5α. These studies further define the role of rhTRIM5α in cell-free and cell-associated HIV transmission and delineate the utility of rhTRIM5α in anti-HIV therapy.     

Active and selective transcytosis of cell-free human immunodeficiency virus through a tight polarized monolayer of human endometrial cells

We report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus.     

Similar regulation of cell surface human T-cell leukemia virus type 1 (HTLV-1) surface binding proteins in cells highly and poorly transduced by HTLV-1-pseudotyped virions

Little is known about the requirements for human T-cell leukemia virus type 1 (HTLV-1) entry, including the identity of the cellular receptor(s). Previous studies have shown that although the HTLV receptor(s) are widely expressed on cell lines of various cell types from different species, cell lines differ dramatically in their susceptibility to HTLV-Env-mediated fusion. Human cells (293, HeLa, and primary CD4(+) T cells) showed higher levels of binding at saturation than rodent (NIH 3T3 and NRK) cells to an HTLV-1 SU immunoadhesin. A direct comparison of the binding of the HTLV-1 surface glycoprotein (SU) immunoadhesin and transduction by HTLV-1 pseudotyped virus revealed parallels between the level of binding and the titer for various cell lines. When cells were treated with phorbol myristate acetate (PMA), which down-modulates a number of cell surface molecules, the level of SU binding was markedly reduced. However, PMA treatment only slightly reduced the titer of murine leukemia virus(HTLV-1) on both highly susceptible and poorly susceptible cells. Treatment of target cells with trypsin greatly reduced binding, indicating that the majority of HTLV SU binding is to proteins. Polycations, which enhance the infectivity of several other retroviruses, inhibited HTLV-1 Env-mediated binding and entry on both human and rodent cells. These results suggest that factors other than the number of primary binding receptors are responsible for the differences in the titers of HTLV-1 pseudotypes between highly susceptible cells and poorly susceptible cells.     

Multiploid inheritance of HIV-1 during cell-to-cell infection

During cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), many viral particles can be simultaneously transferred from infected to uninfected CD4 T cells through structures called virological synapses (VS). Here we directly examine how cell-free and cell-to-cell infections differ from infections initiated with cell-free virus in the number of genetic copies that are transmitted from one generation to the next, i.e., the genetic inheritance. Following exposure to HIV-1-expressing cells, we show that target cells with high viral uptake are much more likely to become infected. Using T cells that coexpress distinct fluorescent HIV-1 variants, we show that multiple copies of HIV-1 can be cotransmitted across a single VS. In contrast to cell-free HIV-1 infection, which titrates with Poisson statistics, the titration of cell-associated HIV-1 to low rates of overall infection generates a constant fraction of the newly infected cells that are cofluorescent. Triple infection was also readily detected when cells expressing three fluorescent viruses were used as donor cells. A computational model and a statistical model are presented to estimate the degree to which cofluorescence underestimates coinfection frequency. Lastly, direct detection of HIV-1 proviruses using fluorescence in situ hybridization confirmed that significantly more HIV-1 DNA copies are found in primary T cells infected with cell-associated virus than in those infected with cell-free virus. Together, the data suggest that multiploid inheritance is common during cell-to-cell HIV-1 infection. From this study, we suggest that cell-to-cell infection may explain the high copy numbers of proviruses found in infected cells in vivo and may provide a mechanism through which HIV preserves sequence heterogeneity in viral quasispecies through genetic complementation.     

Measles virus exits human airway epithelia within dislodged metabolically active infectious centers

Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3–5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2’-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV’s strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.     

Attachment characteristics of normal human cells and virus-infected cells on microcarriers

The attachment kinetics of normal and virus-infected LuMA cells were studied to improve the production of live attenuated varicella viruses in human embryonic lung (LuMA) cells. Normal LuMA cells and LuMA cells infected by varicella virus at various cytopathic effects (CPE) were grown on microcarriers. Ninety-three percent of suspended LuMA cells attached to the solid surface microcarriers within fifteen minutes and cell viability was greater than 95% when the cell suspension was stirred. Low serum levels did not affect the attachment rate of virus-infected cells in the microcarrier culture system. Kinetic studies showed that varicella infected cells had a lower attachment rate than normal LuMA cells. Virus inoculum (= infected cells) at low CPE showed a relatively better attachment rate on cell-laden microcarriers than virus inoculum at a higher CPE. Maximum titers were obtained at 2 days post-infection. Based on cell densities, the use of viral inoculum showing a 40% CPE led to an approximately 2- and 1.2-fold increase in the cell associated and in cell free viruses, respectively, than a virus inoculum with a CPE of 10%.However, the ratio of cell-free to cell-associated virus in a microcarrier culture was very low, approximately0.04–0.06. These studies demonstrate that the virus inoculum resulting in a high CPE yielded a high production of cell-associated and cell-free virus in microcarrier cultures because of the high cellular affinity of the varicella virus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus

Ross River virus (RRV) and Semliki Forest virus (SFV) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. We show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors. Both RRV and SFV glycoproteins considerably expand the host range of the lentivirus vector, and vectors can be efficiently concentrated by ultracentrifugation. A systematic analysis comparing the alphaviral glycoproteins to the vesicular stomatitis virus glycoprotein (VSV-G) revealed that lentivirus vectors incorporate RRV glycoproteins with an efficiency comparable to that of VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments. Together, our data suggest that RRV and SFV glycoproteins might be suitable as alternatives to VSV-G for pseudotyping lentivirus vectors.     

Superinfection of a chimpanzee with a second strain of human immunodeficiency virus.

Two chimpanzees, one (C-499) infected with the acquired immunodeficiency syndrome-associated retrovirus type 2 (ARV-2) strain of human immunodeficiency virus (HIV) and one (C-560) infected with the lymphadenopathy-associated virus type 1 (LAV-1) strain of HIV, were inoculated with approximately 10(4) tissue culture infective doses of the reciprocal strain. At the time of the second inoculation, both chimpanzees had high titers of HIV-specific antibodies, including antibodies that neutralized both virus strains. After inoculation of the second strain of HIV, the antibody titers in both chimpanzees increased 4- to 10-fold, and in one chimpanzee (C-499), the numbers of infectious peripheral blood mononuclear cells (PBMC) increased 1,000-fold to levels that are comparable with those observed after primary HIV infections. By restriction enzyme analysis of virus recovered from PBMC, both ARV-2 and LAV-1 were identified in C-499, thus demonstrating that superinfection had occurred.     

Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus

Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 x 10(5) infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.     

Role of O-glycosylation and expression of CD43 and CD45 on the surfaces of effector T cells in human T cell leukemia virus type 1 cell-to-cell infection

We used replication-dependent retroviral vectors to identify cell surface antigens involved in the cell-to-cell transmission of human T cell leukemia virus type 1 (HTLV-1). We generated monoclonal antibodies (MAbs) against Jurkat T cells and selected several IgM MAbs that strongly inhibited HTLV-1 but not human immune deficiency virus type 1 (HIV-1) cell-to-cell infection. These MAbs recognized the so-called Tn antigen (GalNAcα1-O-Ser/Thr) that arises on Jurkat cells from a mutation in the T-synthase-specific chaperone Cosmc and the consequent loss of O-glycan elongation. Anti-Tn MAbs precipitated two major O-glycan carrier proteins, CD43 and CD45, and caused a strong aggregation of Jurkat cells. The restoration of O-glycosylation in Jurkat cells by stably transducing the wild-type Cosmc gene resulted in a 3- to 4-fold increase in the level of surface expression of CD43 and enhanced HTLV-1 transmission 10-fold in comparison to that of parental cells. The short hairpin RNA (shRNA) knockdown of CD43 or CD45 expression in Jurkat-Cosmc, HBP-ALL, and CEM T cells decreased HTLV-1 infection severalfold. The knockdown of CD45 in Jurkat cells severely reduced both HTLV-1 and HIV-1 infections, but Cosmc coexpression partially rescued infection. HTLV-1 proteins, which assembled in small patches on Jurkat cells, formed large clusters on the surface of Jurkat-Cosmc cells. These data indicate that large aggregates of HTLV-1 assemblies are more infectious than multiple clustered virions. We suggest that heavily O-glycosylated CD43 and CD45 molecules render cells less adhesive, prevent inappropriate cell-cell contacts, and favor the assembly of HTLV-1 particles into large, highly infectious structures on the surface of T cells.     

Characterization of Ebola Virus Entry by Using Pseudotyped Viruses: Identification of Receptor-Deficient Cell Lines

Studies analyzing Ebola virus replication have been severely hampered by the extreme pathogenicity of this virus. To permit analysis of the host range and function of the Ebola virus glycoprotein (Ebo-GP), we have developed a system for pseudotyping these glycoproteins into murine leukemia virus (MLV). This pseudotyped virus, MLV(Ebola), can be readily concentrated to titers which exceed 5 × 10⁶ infectious units/ml and is effectively neutralized by antibodies specific for Ebo-GP. Analysis of MLV(Ebola) infection revealed that the host range conferred by Ebo-GP is very broad, extending to cells of a variety of species. Notably, all lymphoid cell lines tested were completely resistant to infection; we speculate that this is due to the absence of a cellular receptor for Ebo-GP on B and T cells. The generation of high-titer MLV(Ebola) pseudotypes will be useful for the analysis of immune responses to Ebola virus infection, development of neutralizing antibodies, analysis of glycoprotein function, and isolation of the cellular receptor(s) for the Ebola virus.     

Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

Deoxypyrimidine kinase (deoxythymidine [TdR] kinase and deoxycytidine kinase) activity was induced in human embryonic lung cells after infection with varicella-zoster virus (VZ virus). Increased enzyme activity was also produced by using cell-associated virus as inoculum instead of cell-free virus. Anti-VZ virus serum inhibited both the appearance of cytopathic effect and the induction of enzyme activity. The induced TdR kinase activity was more thermostable than that induced by herpes simplex virus type 1. Also, the TdR kinase activity of VZ virus-infected cells was inhibited by dTTP less than in mock-infected cells and more than in herpes simplex virus type 1-infected cells.