A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTR(FLAG)-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 µg SUMO*-CFTR(FLAG)-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.

HIV-1 capsid-targeting domain of cleavage and polyadenylation specificity factor 6.

The antiviral factor CPSF6-358 restricts human immunodeficiency virus type 1 (HIV-1) infection through an interaction with capsid (CA), preventing virus nuclear entry and integration. HIV-1 acquires resistance to CPSF6-358 through an N74D mutation of CA that impairs binding of the antiviral factor. Here we examined the determinants within CPSF6-358 that are necessary for CA-specific interaction. Residues 314 to 322 include amino acids that are essential for CPSF6-358 restriction of HIV-1. Fusion of CPSF6 residues 301 to 358 to rhesus TRIM5α is also sufficient to restrict wild-type but not N74D HIV-1. Restriction is lost if CPSF6 residues in the amino acid 314 to 322 interaction motif are mutated. Examination of the CA targeting motif in CPSF6-358 did not reveal evidence of positive selection. Given the sensitivity of different primate lentiviruses to CPSF6-358 and apparent conservation of this interaction, our data suggest that CPSF6-358-mediated targeting of HIV-1 could provide a broadly effective antiviral strategy.

Flexible use of nuclear import pathways by HIV-1.

HIV-1 replication requires transport of nascent viral DNA and associated virion proteins, the retroviral preintegration complex (PIC), into the nucleus. Too large for passive diffusion through nuclear pore complexes (NPCs), PICs use cellular nuclear transport mechanisms and nucleoporins (NUPs), the NPC components that permit selective nuclear-cytoplasmic exchange, but the details remain unclear. Here we identify a fragment of the cleavage and polyadenylation factor 6, CPSF6, as a potent inhibitor of HIV-1 infection. When enriched in the cytoplasm, CPSF6 prevents HIV-1 nuclear entry by targeting the viral capsid (CA). HIV-1 harboring the N74D mutation in CA fails to interact with CPSF6 and evades the nuclear import restriction. Interestingly, whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics feline immunodeficiency virus nuclear import requirements and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs.

Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

Exogenous retroviruses are obligate cellular parasites that co-opt a number of host proteins and functions to enable their replication and spread. Several host factors that restrict HIV and other retroviral infections have also recently been described. Here we demonstrate that Mov10, a protein associated with P-bodies that has a putative RNA-helicase domain, when overexpressed in cells can inhibit the production of infectious retroviruses. Interestingly, reducing the endogenous Mov10 levels in virus-producing cells through siRNA treatment also modestly suppresses HIV infectivity. The actions of Mov10 are not limited to HIV, however, as ectopic expression of Mov10 restricts the production of other lentiviruses as well as the gammaretrovirus, murine leukemia virus. We found that HIV produced in the presence of high levels of Mov10 is restricted at the pre-reverse transcription stage in target cells. Finally, we show that either helicase mutation or truncation of the C-terminal half of Mov10, where a putative RNA-helicase domain is located, maintained most of its HIV inhibition; whereas removing the N-terminal half of Mov10 completely abolished its activity on HIV. Together these results suggest that Mov10 could be required during the lentiviral lifecycle and that its perturbation disrupts generation of infectious viral particles. Because Mov10 is implicated as part of the P-body complex, these findings point to the potential role of cytoplasmic RNA processing machinery in infectious retroviral production.

The LEM domain proteins emerin and LAP2alpha are dispensable for human immunodeficiency virus type 1 and murine leukemia virus infections.

The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.

HIV-1 protease dimer interface mutations that compensate for viral reverse transcriptase instability in infectious virions.

Mature enzymes encoded within the human immunodeficiency virus type 1 (HIV-1) genome (protease (PR), reverse transcriptase (RT) and integrase (IN)) derive from proteolytic processing of a large polyprotein (Gag-Pol). Gag-Pol processing is catalyzed by the viral PR, which is active as a homodimer. The HIV-1 RT functions as a heterodimer (p66/p51) composed of subunits of 560 and 440 amino acid residues, respectively. Both subunits have identical amino acid sequence, but p51 lacks 120 residues that are removed by the HIV-1 PR during viral maturation. While p66 is the catalytic subunit, p51 has a primarily structural role. Amino acid substitutions affecting the stability of p66/p51 (i.e. F130W) have a deleterious effect on viral fitness. Previously, we showed that the effects of F130W are mediated by p51 and can be compensated by mutation T58S. While studying the dynamics of emergence of the compensatory mutation, we observed that mutations in the viral PR-coding region were selected in HIV clones containing the RT substitution F130W, before the imposition of T58S/F130W mutations. The PR mutations identified (G94S and T96S) improved the replication capacity of the F130W mutant virus. By using a trans-complementation assay, we demonstrate that the loss of p66/p51 heterodimer stability caused by Trp130 can be attributed to an increased susceptibility of RT to viral PR degradation. Recombinant HIV-1 PRs bearing mutations G94S or T96S showed decreased dimer stability and reduced catalytic efficiency. These results were consistent with crystallographic data showing the location of both residues in the PR dimerization interface.

Analysis of amino acids in the beta7-beta8 loop of human immunodeficiency virus type 1 reverse transcriptase for their role in virus replication.

The HIV-1 p51/p66 reverse transcriptase (RT) heterodimer interface comprises, in part, intermolecular interaction of the loop region between beta-strands 7 and 8 (beta7-beta8 loop) in the p51 fingers subdomain with the p66 palm subdomain. In this study, for the first time in the context of infectious HIV-1 particles, we analyzed the contribution of amino acid residues (S134, I135, N136, N137, T139 and P140) in the beta7-beta8 loop for RT heterodimerization, enzymatic activity, and virus infectivity. Mutating asparagine 136 to alanine (N136A) reduced viral infectivity and enzyme activity dramatically. The N136A mutation appeared to destabilize the RT heterodimer and render both the p66 and p51 subunits susceptible to aberrant cleavage by the viral protease. Subunit-specific mutagenesis demonstrated that the presence of the N136A mutation in the p51 subunit alone was sufficient to cause degradation of RT within the virus particle. Alanine mutation at other residues of the beta7-beta8 loop did not affect either RT stability or virus infectivity significantly. None of the beta7-beta8 loop alanine mutations affected the sensitivity of virus to inhibition by NNRTIs. In the context of infectious virions, our results indicate a critical role of the p51 N136 residue within the beta7-beta8 loop for RT heterodimer stability and function. These findings suggest the interface comprising N136 in p51 and interacting residues in p66 as a possible target for rational drug design.

Tethering KSRP, a decay-promoting AU-rich element-binding protein, to mRNAs elicits mRNA decay.

Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3' untranslated regions that act as mRNA stability determinants by interacting with ARE-binding proteins (ARE-BPs). We have destabilized two mRNAs by fusing sequence-specific RNA-binding proteins to KSRP, a decay-promoting ARE-BP, in a tethering assay. These results support a model that KSRP recruits mRNA decay machinery/factors to elicit decay. The ability of tethered KSRP to elicit mRNA decay depends on functions of known mRNA decay enzymes. By targeting the Rev response element of human immunodeficiency virus type 1 by using Rev-KSRP fusion protein, we degraded viral mRNA, resulting in a dramatic reduction of viral replication. These results provide a foundation for the development of novel therapeutic strategies to inhibit specific gene expression in patients with acquired or hereditary diseases.

Analysis of human immunodeficiency virus type 1 reverse transcriptase subunit structure/function in the context of infectious virions and human target cells.

The reverse transcriptase (RT) of all retroviruses is required for synthesis of the viral DNA genome. The human immunodeficiency virus type 1 (HIV-1) RT exists as a heterodimer made up of 51-kDa and 66-kDa subunits. The crystal structure and in vitro biochemical analyses indicate that the p66 subunit of RT is primarily responsible for the enzyme's polymerase and RNase H activities. Since both the p51 and p66 subunits are generated from the same coding region, as part of the Pr160(Gag-Pol) precursor protein, there are inherent limitations for studying subunit-specific function with intact provirus in a virologically relevant context. Our lab has recently described a novel system for studying the RT heterodimer (p51/p66) wherein a LTR-vpr-p51-IRES-p66 expression cassette provided in trans to an RT-deleted HIV-1 genome allows precise molecular analysis of the RT heterodimer. In this report, we describe in detail the specific approaches, alternative strategies, and pitfalls that may affect the application of this novel assay for analyzing RT subunit structure/function in infectious virions and human target cells. The ability to study HIV-1 RT subunit structure/function in a physiologically relevant context will advance our understanding of both RT and the process of reverse transcription. The study of antiretroviral drugs in a subunit-specific virologic context should provide new insights into drug resistance and viral fitness. Finally, we anticipate that this approach will help elucidate determinants that mediate p51-p66 subunit interactions, which is essential for structure-based drug design targeting RT heterodimerization.

Identification of amino acid residues in the human immunodeficiency virus type-1 reverse transcriptase tryptophan-repeat motif that are required for subunit interaction using infectious virions.

The human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) functions as a heterodimer (p51/p66), which makes disruption of subunit interactions a possible target for antiviral drug design. Our understanding of subunit interface interactions has been limited by the lack of virus-based approaches for studying the heterodimer. Therefore, we developed a novel subunit-specific mutagenesis approach that enables precise molecular analysis of the heterodimer in the context of infectious HIV-1 particles. Here, we analyzed the contributions of amino acid residues comprising the Trp-motif to RT subunit interaction and function. Our results reveal important inter- and intra-subunit interactions of residues in the Trp-motif. A tryptophan cluster in p51 (W398, W402, W406, W414), proximal to the interface, was found to be important for p51/p66 interaction and stability. At the dimer interface, residues W401, Y405 and N363 in p51 and W410 in p66 mediate inter-subunit interactions. The W401 residue is critical for RT dimerization, exerting distinct effects in p51 and p66. Our analysis of the RT heterodimerization enhancing non-nucleoside RT inhibitor (NNRTI), efavirenz, indicates that the effects of drugs on RT dimer stability can be examined in human cells. Thus, we provide the first description of subunit-specific molecular interactions that affect RT heterodimer function and virus infection in vivo. Moreover, with heightened interest in novel RT inhibitors that affect dimerization, we demonstrate the ability to assess the effects of RT inhibitors on subunit interactions in a physiologically relevant context.

Subunit-specific analysis of the human immunodeficiency virus type 1 reverse transcriptase in vivo.

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (p51 and p66). Since p51 and p66 are derived from the same coding region, subunit-specific structure-function studies of RT have been conducted exclusively by in vitro biochemical approaches. To study RT subunit function in the context of infectious virus, we constructed an LTR-vpr-p51-IRES-p66 expression cassette in which the HIV-1 vpr gene was fused in frame with p51, followed by an internal ribosome entry site (IRES) sequence and the p66 coding region. By coexpression with RT-deficient proviral DNA, we demonstrated that the p66 subunit is specifically and selectively packaged into virions as a Vpr-p51/p66 complex. Our analysis showed that cleavage by the viral protease liberates Vpr and generates functional heterodimeric RT (p51/p66) that supports HIV-1 reverse transcription and virus infection. By exploiting this novel trans-complementation approach, we demonstrated, for the first time with infectious virions, that the YMDD aspartates of p66 are both required and sufficient for RT polymerase function. Mutational analyses of the p51 YMDD aspartates indicated that they play an important structural role in p51 folding and subunit interactions that are required for the formation of an active RT heterodimer within infected cells. Understanding the role of the individual RT subunits in RNA- and DNA-dependent DNA synthesis is integral to our understanding of RT function. Our findings will lead to important new insights into the role of the p51 and p66 subunits in HIV-1 reverse transcription.