Chemically inactivated adenoviral vectors that can efficiently transduce target cells when delivered in the form of virus-microbead conjugates.

Safe and effective use of viral vectors for gene therapeutics requires versatile control over their delivery to target sites in human subjects. We have developed a strategy for the creation of adenoviral vectors that possess conditional infectivity. The adenoviral vectors used were inactivated chemically such that they had little or no ability to infect cells. However, when such chemically inactivated adenoviral vectors were conjugated to the surfaces of appropriate microbeads and the resulting adenovirus-microbead conjugates were provided with the ability to associate stably with cells, the infectivity of these adenoviral vectors was restored. For certain target cell lines, the infectivity of such adenovirus-microbead conjugates became even higher than that of free, unmodified adenoviral vectors. As a result of the chemical inactivation of viral infectivity, any adenoviral particles that become free from the microbeads should be noninfectious. Thus, these adenoviral vectors have an infectivity that is conditional: They can only infect cells, to which their microbead conjugates come into stable contact. These results lay the groundwork for the creation of targetable adenovirus-microbead conjugates with greater efficacy and safety as delivery agents for gene therapeutics.

Photoactivatable retroviral vectors: a strategy for targeted gene delivery.

We have explored a novel strategy for the targeting of retroviral vectors to particular sites or cell types. This strategy involves a method whereby the infectivity of a retroviral vector is neutralized by treatment of viral particles with a photocleavable, biotinylation reagent. These modified viral vectors possess little to no infectivity for target cells. Exposure of these modified viral vectors to long-wavelength UV light induces a reversal of the neutralizing, chemical modification resulting in restoration of infectivity to the viral vector. This infectivity 'trigger' possesses great potential, both as a research tool and as a novel tactic for the targeting of gene-transfer agents, since it would become possible to direct both the time and location of a viral infection in a versatile manner.

Analysis of the SH3-binding region of HIV-1 nef: partial functional defects introduced by mutations in the polyproline helix and the hydrophobic pocket.

An SH3-binding domain within the Nef protein of primate lentiviruses has been reported to be important to viral replication and infectivity and dispensable for CD4 downregulation, but its precise role remains unclear. This study investigates the effects of mutations in both the polyproline helix and in the hydrophobic pocket that constitute the SH3-binding domain of Nef. The data demonstrate that the well-studied mutation of the central prolines is only partially disruptive to viral infectivity and replication. The central prolines also make a subtle contribution to the efficiency of CD4 downregulation, detectable only using low levels of Nef expression. Mutation of a conserved arginine in the polyproline helix abrogated more completely Nef-mediated enhancement of viral infectivity; this mutation also adversely affected CD4 downregulation at low levels of Nef expression. Only the R77A mutation substantially impaired downregulation of class I MHC. However, mutation of the central prolines and of R77 yielded proteins that were expressed less efficiently than wild-type Nef. The R77A mutant was expressed most poorly, compatible with its defective phenotypes in all assays. Mutations of the hydrophobic pocket were minimally detrimental to both the virologic and the receptor modulatory functions of Nef. Taken together, this analysis suggests that mutations in the SH3-binding domain do not abrogate fully any Nef-associated phenotype in the absence of detrimental effects on protein expression. We suggest that mutations in this domain can introduce incomplete effects caused by subtle impairments to protein expression; these effects may appear selective under certain experimental conditions due to different sensitivities of the assays to the level of Nef expression.

The dileucine-based sorting motif in HIV-1 Nef is not required for down-regulation of class I MHC.

A dileucine-based protein sorting motif has recently been identified within the C-terminal, solvent-exposed loop of HIV-1 Nef and has been shown to be required for Nef-mediated down-regulation of CD4 and for optimal viral infectivity. Here, we report that mutation of the dileucine motif has no effect on Nef-mediated down-regulation of class I MHC heavy chain. Instead, deletion of an acidic domain just N-terminal of the polyproline helix of the SH3-binding domain significantly impairs this function. These data indicate that down-regulation of class I MHC and CD4 are mechanistically distinct processes. The data also suggest that protein interactions mediated by the acidic domain, rather than by the dileucine motif, may contribute to this function of Nef.

Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity.

The HIV-1 Nef protein is important for pathogenesis, enhances viral infectivity, and regulates the sorting of at least two cellular transmembrane proteins, CD4 and major histocompatibility complex (MHC) class I. Although several lines of evidence support the hypothesis that the Nef protein interacts directly with the cellular protein sorting machinery, the sorting signal in HIV-1 Nef has not been identified. By using a competition assay that functionally discriminates between dileucine-based and tyrosine-based sorting signals, we have categorized the motif through which Nef interacts with the sorting machinery as dileucine-based. Inspection of diverse Nef proteins from HIV-1, HIV-2, and simian immunodeficiency virus revealed a well-conserved sequence in the central region of the C-terminal, solvent-exposed loop of Nef (E/DXXXLphi) that conforms to the consensus sequence of the dileucine-based sorting motifs found in cellular transmembrane proteins. This sequence in NefNL4-3, ENTSLL, functioned as an endocytosis signal when appended to the cytoplasmic tail of a heterologous protein. The leucine residues in this motif were required for the interaction of full-length Nef with the dileucine-based sorting pathway and were required for Nef-mediated down-regulation of CD4. These leucine residues were also required for optimal viral infectivity. These data indicate that a dileucine-based sorting signal in Nef is utilized to address the cellular sorting machinery. The data also suggest that an influence on the distribution of cellular transmembrane proteins may mechanistically unite two previously distinct properties of Nef: down-regulation of CD4 and enhancement of viral infectivity.

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.

Recombinant core streptavidins. A minimum-sized core streptavidin has enhanced structural stability and higher accessibility to biotinylated macromolecules.

Two recombinant core streptavidins were designed and characterized to understand the role of the terminal sequences, present in naturally truncated core streptavidins, in the properties of streptavidin. One recombinant core streptavidin, Stv-25, has an amino acid sequence very similar to natural core streptavidins. The other recombinant molecule, Stv-13, has further truncation of the terminal residues and consists essentially of only the beta-barrel structure characteristic of streptavidin. These recombinant core streptavidins are tetrameric and bind four biotins/molecule, as does natural streptavidin. The solubility characteristics of Stv-13, determined by varying the concentration of ammonium sulfate or ethanol, were almost the same as those of Stv-25 and natural core streptavidin. However, Stv-13 showed an enhanced structural stability compared with Stv-25 and natural core streptavidin. For example, Stv-13 retained greater than 80% of its biotin binding ability after incubation in 6 M guanidine hydrochloride at pH 1.5, under which conditions, Stv-25 and natural core streptavidin retained only about 20% of their biotin binding ability. In addition, Stv-13 showed higher accessibility to biotinylated DNA than natural core streptavidin. Apparently, the terminal regions, present on the surface of natural core streptavidin, spatially hinder biotinylated macromolecules from approaching the biotin binding sites.

The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation.

Recent data on the phenotype of nef-defective HIV-1 in vitro indicate a new function of the Nef gene product: enhancement of viral infectivity. Single-cycle replication studies have suggested that Nef enhances the efficiency of an early step during viral replication, a step that leads to the establishment of viral DNA. To test this interpretation, the accumulation of low-molecular-weight (unintegrated) viral DNA was measured in cells following exposure to wild-type and nef-defective viruses. nef-defective virus accumulated less DNA than the wild type. This difference was observed after as little as 5 hr of exposure to virus. However, the reverse transcriptase activities of wild-type and nef-defective viruses were equal when measured in cell-free assays using either exogenous or endogenous templates. In addition, the abilities of these viruses to bind and enter cells were not significantly different. Together, these data suggest that Nef optimizes postentry events that are required for efficient synthesis of viral DNA. To determine if these effects were related to the property of Nef-mediated downregulation of CD4, growth curves of these viruses were determined using cells that express a CD4 molecule unable to respond to Nef. nef-defective virus remained attenuated in these cells, indicating that Nef-mediated downregulation of CD4 is not required for Nef-mediated enhancement of viral propagation in vitro.