Food Insecurity, Chronic Disease and Quality of Life among Deaf Adults who use American Sign Language.

This study examined the relationships of food insecurity, chronic diseases, and QoL in a U.S. sample of 630 deaf adults (18 to 89 years old; 55% female) who used American Sign Language (ASL). Measures of USDA Food Security Module, self-reported diagnoses of chronic diseases, and QoL were administered in ASL and English. Approximately 22% reported facing food insecurity, with low food security (11%) and very low food security (11.4%). QoL, but not the presence of chronic diseases, was significantly associated with food insecurity. Higher income and absence of depression acted as protective factors, reducing the risk for food insecurity.

The roles of cellular factors in retroviral integration.

A key early step in the retroviral life cycle is the integration of reverse-transcribed viral cDNA into a chromosome of an infected cell. The key protein player in retroviral integration is the viral integrase, which enters the cell as part of the virus. Although purified integrase protein is necessary and sufficient to perform the basic catalytic DNA breakage and joining steps of retroviral integration, a variety of normal cellular proteins have been implicated as playing important roles in establishing the integrated provirus in cells. This chapter reviews the roles of host cell factors that function during integrase catalysis, during the repair of the resulting DNA recombination intermediate, and by potentially guiding viral preintegration complexes to their chromosomal locations for cDNA integration. The potential to interfere with proper integration by blocking either integrase catalysis or the function of cellular integration cofactors is also discussed.

Asymmetric processing of human immunodeficiency virus type 1 cDNA in vivo: implications for functional end coupling during the chemical steps of DNA transposition.

Retroviral integration, like all forms of DNA transposition, proceeds through a series of DNA cutting and joining reactions. During transposition, the 3' ends of linear transposon or donor DNA are joined to the 5' phosphates of a double-stranded cut in target DNA. Single-end transposition must be avoided in vivo because such aberrant DNA products would be unstable and the transposon would therefore risk being lost from the cell. To avoid suicidal single-end integration, transposons link the activity of their transposase protein to the combined functionalities of both donor DNA ends. Although previous work suggested that this critical coupling between transposase activity and DNA ends occurred before the initial hydrolysis step of retroviral integration, work in the related Tn10 and V(D)J recombination systems had shown that end coupling regulated transposase activity after the initial hydrolysis step of DNA transposition. Here, we show that integrase efficiently hydrolyzed just the wild-type end of two different single-end mutants of human immunodeficiency virus type 1 in vivo, which, in contrast to previous results, proves that two functional DNA ends are not required to activate integrase's initial hydrolysis activity. Furthermore, despite containing bound protein at their processed DNA ends, these mutant viruses did not efficiently integrate their singly cleaved wild-type end into target DNA in vitro. By comparing our results to those of related DNA recombination systems, we propose the universal model that end coupling regulates transposase activity after the first chemical step of DNA transposition.

Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines.

Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

Both the structure and DNA binding function of the barrier-to-autointegration factor contribute to reconstitution of HIV type 1 integration in vitro.

Retroviral integration is mediated by viral preintegration complexes (PICs), and human immunodeficiency virus type 1 (HIV-1) PICs treated with high salt lose their in vitro integration activity. Barrier-to-autointegration factor (BAF) is a host protein that efficiently restores PIC activity, but the mechanism(s) by which BAF participates in HIV-1 integration remains largely unknown. Here we developed a gel shift assay to study BAF DNA binding, and analyzed 14 mutant proteins containing substitutions of conserved residues for binding and PIC reconstitution activities. Although wild-type BAF efficiently bound double-stranded DNA, binding to single-stranded DNA, RNA, or an RNA/DNA hybrid was not detected, suggesting that BAF associates with retroviral cDNA relatively late during reverse transcription. Although some of the BAF mutant proteins efficiently bound DNA, others were defective for binding. Mutants that bound DNA efficiently reconstituted HIV-1 integration, even though in one case binding was just 0.2% of wild-type BAF. Although misfolded mutants did not reconstitute integration, a structurally intact DNA binding-defective mutant displayed partial activity at high BAF concentration. We therefore conclude that both BAF protein structure and its DNA binding activity play roles in reconstituting HIV-1 integration in vitro.

Characterization of a replication-defective human immunodeficiency virus type 1 att site mutant that is blocked after the 3' processing step of retroviral integration.

Two activities of retroviral integrase, 3' processing and DNA strand transfer, are required to integrate viral cDNA into a host cell chromosome. Integrase activity has been analyzed in vitro using purified protein and recombinant DNA substrates that model the U3 and U5 ends of viral cDNA or by using viral preintegration complexes (PICs) that form during virus infection. Numerous studies have investigated changes in integrase or viral DNA for effects on both 3' processing and DNA strand transfer activities using purified protein, but similar analyses have not been carried out using PICs. Here, we analyzed PICs from human immunodeficiency virus type 1 (HIV-1) strain 604del, an integration-defective mutant lacking 26 bp of U5, and revE1, a revertant of 604del containing an additional 19-bp deletion, for levels of 3' processing activity that occurred in infected cells and for levels of in vitro DNA strand transfer activity. Whereas revE1 supported one-third to one-half of the level of wild-type DNA strand transfer activity, the level of 604del DNA strand transfer activity was undetectable. Surprisingly, integrase similarly processed the 3' ends of 604del and revE1 in vivo. We therefore conclude that 604del is blocked in its ability to replicate in cells after the 3' processing step of retroviral integration. Whereas Western blotting showed that wild-type, revE1, and 604del PICs contained similar levels of integrase protein, Mu-mediated PCR footprinting revealed only minimal protein-DNA complex formation at the ends of 604del cDNA. We propose that 604del is replication defective because proteins important for DNA strand transfer activity do not stably associate with this cDNA after in vivo 3' processing by integrase.

Structure-based mutagenesis of the human immunodeficiency virus type 1 DNA attachment site: effects on integration and cDNA synthesis.

Sequences at the ends of linear retroviral cDNA important for integration define the viral DNA attachment (att) site. Whereas determinants of human immunodeficiency virus type 1 (HIV-1) integrase important for replication in T lymphocytes have been extensively characterized, regions of the att site important for viral spread have not been thoroughly examined. Previous transposon-mediated footprinting of preintegration complexes isolated from infected cells revealed enhanced regions of bacteriophage Mu insertion near the ends of HIV-1 cDNA, in the regions of the att sites. Here, we identified the subterminal cDNA sequences cleaved during in vitro footprinting and used this structure-based information together with results of previous work to construct and characterize 24 att site mutant viruses. We found that although subterminal cDNA sequences contributed to HIV-1 replication, the identities of these bases were not critical for integration. In contrast, the phylogenetically conserved CA dinucleotides located at the ends of HIV-1 contributed significantly to virus replication and integration. Mutants containing one intact CA end displayed delays in peak virus growth compared to the wild type. In contrast, double mutant viruses lacking both CAs were replication defective. The A of the CA appeared to be the most critical determinant of integration, because two different U5 mutant viruses containing the substitution of TG for CA partially reverted by changing the G back to A. We also identified a U5 deletion mutant in which the CA played a crucial role in reverse transcription.

In vivo analysis of retroviral integrase structure and function.

There are two retroviral integration loci. One encodes the transacting IN protein, which is cleaved from the carboxyl terminus of the Gag-Pol polyprotein precursor during virus assembly. The second locus is the cis-acting attachment (att) site, comprising the terminal sequences at the U3 and U5 ends of linear viral cDNA. Integrase and att site mutant viruses can be blocked at different steps of the viral replication cycle. Class I IN mutants are blocked specifically at the integration step. Class II IN mutants, on the other hand, display pleiotropic defects, most notably in virion morphogenesis and/or reverse transcription. Mutations in the U5 end att site can also disrupt reverse transcription in addition to integration. It is prudent to use caution when interpreting results of in vivo mutagenesis experiments that target retroviral IN and the att site.

Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome.

Mu-mediated polymerase chain reaction footprinting was used to investigate the protein-DNA structure of human immunodeficiency virus type I (HIV-I) preintegration complexes. Preintegration complexes were partially purified from cells after using an established coculture infection technique as well as a novel technique using cell-free supernatant from transfected cells as the source of virus. Footprinting revealed that bound proteins protected the terminal 200-250 base pairs of each viral end from nuclease attack. Bound proteins also caused strong transpositional enhancements near each end of HIV-I. In contrast, regions of viral DNA internal to the ends did not show evidence of strong protein binding. The end regions of preintegrative HIV-I apparently form a unique nucleoprotein structure, which we term the intasome to distinguish it from the greater preintegration complex. Our novel system also allowed us to analyze the structure and function of preintegration complexes isolated from cells infected with integrase mutant viruses. Complexes were derived from viruses defective for either integrase catalysis, integrase binding to the viral DNA substrate, or an unknown function in the carboxyl-terminal domain of the integrase protein. None of these mutant complexes supported detectable integration activity. Despite the presence of the mutant integrase proteins in purified samples, none of these nucleoprotein complexes displayed the native intasome structure detected in wild-type preintegration complexes. We conclude that multiple integrase functions are required to form the native structure of the HIV-I intasome in infected cells.

The barrier-to-autointegration protein is a host factor for HIV type 1 integration.

In vivo, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display in vitro integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein-DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5-20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5-10 microM) human host factor HMG I(Y) was required. Similarly high levels (3-8 microM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein-DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.

A mutation in integrase can compensate for mutations in the simian immunodeficiency virus att site.

Sequences at the left terminus of U3 in the left long terminal repeat (LTR) and at the right terminus of U5 in the right LTR are important for integration of retroviral DNA. In the infectious pathogenic molecular clone of simian immunodeficiency virus strain mac239 (SIVmac239), 10 of the 12 terminal base pairs form an imperfect inverted repeat structure (5' TGGAAGGGATTT 3' [nucleotides 1 to 12] and 3' ACGATCCCTAAA 5' [nucleotides 10279 to 10268]). Nineteen different mutant forms of SIVmac239 proviral DNA with changes at one or more of the positions in each of the 12-terminal-base-pair regions were constructed. Viral replication was severely or completely compromised with nine of these mutants. Revertants appeared 40 to 50 days after transfection in two independent experiments with mutant 7, which contained changes of AGG to TAC at positions 5 to 7 in U3 and TCC to GAA at positions 10275 to 10273 in U5. Virus produced at these times from mutant 7 transfection replicated upon reinfection with only a slight delay when compared to the wild type. Sequence analysis of the LTR and integrase regions from infected cultures revealed two predominant changes: G to A at position 10275 in U5 and Glu to Lys at position 136 in integrase. Derivatives of clone 7 in which these changes were introduced individually and together were constructed by site-specific mutagenesis. Each change individually restored replication capacity only partially. However, the combination of both mutations restored replicative capacity to that of the original revertants. These results indicate that changes in integrase can compensate for mutations in the terminal nucleotides of the SIV LTR. The results further indicate that resistance to integrase inhibitors may include both integrase and LTR mutations.

Structure-based mutagenesis of the catalytic domain of human immunodeficiency virus type 1 integrase.

Two different crystal structures of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalytic domain were analyzed for interactions at the enzyme active site. Gln-62 and Glu-92 interact with active-site residue Asp-64, and Lys-136 interacts with active-site residue Asp-116 across a dimer interface. Conservative and nonconservative substitutions were introduced at these positions to probe the roles of these interactions in HIV-1 integration. Purified mutant proteins were assayed for in vitro 3' processing, DNA strand transfer, and disintegration activities, and HIV-1 mutants were assayed for virion protein composition, reverse transcription, and infectivities in human cell lines. Each of the mutant IN proteins displayed wild-type disintegration activity, indicating that none of the interactions is essential for catalysis. Mutants carrying Gln or Ala for Glu-92 displayed wild-type activities, but substituting Lys for Glu-92 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold and yielded a replication-defective IN active-site mutant viral phenotype. Substituting Glu for Gln-62 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold without grossly affecting viral replication kinetics, suggesting that HIV-1 can replicate in T-cell lines with less than the wild-type level of IN activity. The relationship between IN solubility and HIV-1 replication was also investigated. We previously showed that substituting Lys for Phe-185 dramatically increased the solubility of recombinant IN but caused an HIV-1 particle assembly defect. Mutants carrying His at this position displayed increased solubility and wild-type replication kinetics, showing that increased IN solubility per se is not detrimental to virus growth.

Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking.

Analysis of the crystal structure of HIV-1 integrase reveals a cluster of lysine residues near the active site. Using site-directed mutagenesis and photo-crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159 to glutamate led to a loss of both 3' processing and strand transfer activities in vitro while maintaining the ability to interact with nonspecific DNA and support disintegration. However, mutation of both residues to glutamate produced a synergistic effect eliminating nearly all nonspecific DNA interaction and disintegration activity. In addition, virus containing either of these changes was replication-defective at the step of integration. Photo-crosslinking, using 5-iododeoxyuracil-substituted oligonucleotides, suggests that Lys159 interacts at the N7 position of the conserved deoxyadenosine adjacent to the scissile phosphodiester bond of viral DNA. Sequence conservation throughout retroviral integrases and certain bacterial transposases (e.g. Tn10/IS10) supports the premise that within those families of polynucleotidyl transferases, these residues are strategic for DNA interaction.

Reversion of a human immunodeficiency virus type 1 integrase mutant at a second site restores enzyme function and virus infectivity.

The integration of a DNA copy of the retroviral RNA genome into the host cell genome is essential for viral replication. The virion-associated integrase protein, encoded by the 3' end of the viral pol gene, is required for integration. Stable virus-producing T-cell lines were established for replication-defective human immunodeficiency virus type 1 carrying single amino acid substitutions at conserved residues in the catalytic domain of integrase. Phenotypically reverted virus was detected 12 weeks after transfection with the integrase mutant carrying the P-109-->S mutation (P109S). Unlike the defective P109S virus, the revertant virus (designated P109SR) grew in CD4+ SupT1 cells. In addition to the Ser substitution at Pro-109, P109SR had a second substitution of Ala for Thr at position 125 in integrase. Site-directed mutagenesis was used to show that the P109S T125A genotype was responsible for the P109SR replication phenotype. The T125A substitution also rescued the in vitro enzyme activities of recombinant P109S integrase protein. P109S integrase did not display detectable 3' processing or DNA strand transfer activity, although 5 to 10% of wild-type disintegration activity was detected. P109S T125A integrase displayed nearly wild-type levels of 3' processing, DNA strand transfer, and disintegration activities, confirming that T125A is a second-site intragenic suppressor of P109S. P109S integrase ran as a large aggregate on a size exclusion column, whereas wild-type integrase ran as a monomer and P109S T125A integrase ran as a mixed population. Pro-109 and Thr-125 are not immediately adjacent in the crystal structure of the integrase catalytic domain. We suggest that the T125A substitution restores integrase function by stabilizing a structural alteration(s) induced by the P109S mutation.

Biochemical characterization of recombinant equine infectious anemia virus integrase.

The integrase from equine infectious anemia virus (ELAV) was expressed in Escherichia coli as a polyhistidine fusion protein. The protein was purified under native and denaturing conditions using one-step nickel-affinity chromatography. The purified denatured protein was refolded in the presence of detergent. In vitro 3' processing and DNA strand transfer activities were analyzed under Mg(2+)- and Mn(2+)-dependent reaction conditions. Both protein preparations were similarly active. Only one viral DNA end was efficiently integrated during Mn(2+)-and Mg(2+)-dependent DNA strand transfer. Water was the predominant nucleophile for Mg(2+)- and Mn(2+)-dependent 3' processing activity. The results underscore functional similarities between EIAV integrase and the previously characterized HIV-1 enzyme.

A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in multimerization.

Structural studies of human immunodeficiency virus type 1 (HIV-1) integrase have been impeded by the low solubility of the protein. By systematic replacement of hydrophobic residues, we previously identified a single amino acid change (F185K) that dramatically improved the solubility of the catalytic domain of HIV-1 integrase and enabled the structure to be determined by x-ray crystallography. We have introduced the same mutation into full-length HIV-1 integrase. The resulting recombinant protein is soluble and fully active in vitro, whereas, HIV-1 carrying the mutation is replication-defective due to improper virus assembly. Analysis of the recombinant protein by gel filtration and sedimentation equilibrium demonstrate a dimer-tetramer self-association. We find that the regions involved in multimerization map to both the catalytic core and carboxyl-terminal domains. The dramatically improved solubility of this protein make it a good candidate for structural studies.

Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity.

The integrase protein from human immunodeficiency virus type 1 (HIV-1) has generally been reported to require Mn2+ for efficient in vitro activity. We have reexamined the divalent metal ion requirements of HIV-1 integrase and find that the protein is capable of promoting efficient 3' processing and DNA strand transfer with either Mn2+ or Mg2+. The metal ion preference depended upon the reaction conditions. HIV-1 integrase displayed significantly less nonspecific nuclease activity in reaction mixtures containing Mg2+ than it did under the previously described reaction conditions with mixtures containing Mn2+.

Solution structure of the DNA binding domain of HIV-1 integrase.

The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site.

Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication.

The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes.

Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1.

Certain human immunodeficiency virus type 1 (HIV-1) isolates are able to productively infect nondividing cells of the monocyte/macrophage lineage. We have used a molecular genetic approach to construct two different HIV-1 integrase mutants that were studied in the context of an infectious, macrophage-tropic HIV-1 molecular clone. One mutant, HIV-1 delta D(35)E, containing a 37-residue deletion within the central, catalytic domain of integrase, was noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages. The HIV-1 delta D(35)E mutant, however, exhibited defects in the assembly and/or release of progeny virions in transient transfection assays, as well as defects in entry and/or viral DNA synthesis during the early stages of monocyte-derived macrophage infection. The second mutant, HIV-1D116N/8, containing a single Asp-to-Asn substitution at the invariant Asp-116 residue of integrase, was also noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages but, in contrast to HIV-1 delta D(35)E, was indistinguishable from wild-type virus in reverse transcriptase production. PCR analysis indicated that HIV-1D116N/8 entered monocyte-derived macrophages efficiently and reverse transcribed its RNA but was unable to complete its replication cycle because of a presumed block to integration. These data are consistent with the hypothesis that integration is an obligate step in productive HIV-1 infection of activated peripheral blood mononuclear cells and primary human macrophage cultures.