Molecular Mechanisms of Gap Junction Regulation by the E3 Ubiquitin Ligase WWP1

Ubiquitylation is a post-translational modification that influences a wide variety of cellular processes including protein degradation, protein subcellular localization, cell cycle progression, transcription, and DNA damage repair. Covalent attachment of the small ubiquitin molecule to a target protein involves the sequential action of three enzymes (E1, E2, and E3). In this process, substrate specificity is conferred by the E3 ligase. Our work has focused on the function of one such E3 ubiquitin ligase, WWP1. Known targets of WWP1 include cell cycle proteins, tumor suppressors, and transcription factors that promote differentiation of mesenchymal stem cells to the osteoid lineage. Recently, we have identified a novel target of WWP1 – the gap junction protein connexin (CX) 43. In particular, we found that mice overexpressing WWP1 had a 90% reduction in CX43 within the myocardium and died from ventricular arrhythmias as a consequence. CX43 is a transmembrane protein that oligomerizes to form intercellular channels which facilitate communication between adjacent cells via the transfer of small molecules. This metabolic and electrical coupling of adjacent cells plays a vital role in almost all cellular processes including growth and differentiation, cell division, and homeostasis as well as in electroconduction of the heart. Therefore, it is not surprising that CX43 is broadly expressed in nearly every cell type, and it is likely that there are commonalities underlying the regulation of CX43 in all cells that express it. Of particular interest is the fact that CX43 has a remarkably short half-life for an integral membrane protein (on the order of 1-5 hours) in all cell types examined. To investigate the molecular mechanisms involved in CX43 turnover, we used an established tissue culture system to examine the effects of changes in expression of WWP1 on the stability and subcellular localization of CX43. We found that CX43 could co-immunoprecipitate with WWP1, and this interaction was dependent on the PPXY motif of CX43. This association promoted the K27- and K29-linked polyubiquitylation of CX43 by WWP1. Co-immunoprecipitation of WWP1 with CX43 and subsequent ubiquitylation of CX43 was enhanced in the presence of phorbol 12-myristate 13-acetate (PMA) which has been reported to induce mitogen activated protein kinase (MAPK)-dependent phosphorylation and subsequent internalization of CX43 from the cell membrane to the early endosomes. WWP1-mediated ubiquitylation was found to destabilize CX43, as the overexpression of wild type WWP1 in HeLa-CX43 cells reduced the half-life of CX43 from 2 hours to less than 1 hour, while a mutant version of WWP1 lacking ubiquitin ligase activity (C866S) had no significant effect on the stability of CX43. The increased turnover of CX43 associated with the overexpression of WWP1 also significantly reduced gap junction-mediated intercellular communication. Further investigation of the role of WWP1-mediated ubiquitylation on CX43 trafficking revealed that the ligase activity of WWP1 promoted trafficking of WWP1 from the early endosome to the late endosome with subsequent delivery to the lysosome for degradation. These observations were corroborated when endogenous WWP1 was knocked down using a siRNA pool that targets WWP1. Specifically, loss of WWP1 was associated with increased levels of CX43 on the plasma membrane and with decreased trafficking of CX43 from the early endosome to the late endosome. Instead, with WWP1 knockdown, increased co-localization of CX43 with the recycling endosome marker RAB11 was noted. These data, in conjunction with our overexpression studies, suggest that WWP1 ubiquitylates CX43 in the early endosome, and this signal is required for trafficking to the lysosome for degradation. In the absence of functional WWP1, CX43 is trafficked back to the plasma membrane via an endogenous recycling pathway whose existence hitherto has been sparsely described in the literature. Collectively, this study has identified a novel role for WWP1-mediated ubiquitylation in the trafficking and lysosomal degradation of CX43 involving an atypical ubiquitin linkage. Gap junction dysregulation is associated numerous pathological conditions including arrhythmia, skin defects, cataracts and carcinogenesis. Therefore, studies like this one that elucidate the molecular mechanisms underlying the regulation of CX43 will greatly contribute towards the development of novel therapeutics

Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs

<p>Abstract</p> <p>Background</p> <p>Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection.</p> <p>Results</p> <p>Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (<it>env</it>) gene as well as a siRNA specific for a downstream target sequence in the subtype D <it>env </it>gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved env regions suggest that other mechanisms are at play.</p> <p>Conclusion</p> <p>These findings show that siRNAs can be used as an efficient in vitro tool for enriching recombinants, to facilitate further study on mechanisms of intersubytpe HIV-1 recombination, and to generate replication-competent intersubtype recombinant proteins with a breadth in HIV-1 diversity for future vaccine studies.</p

Influence of sequence identity and unique breakpoints on the frequency of intersubtype HIV-1 recombination

BACKGROUND: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population. RESULTS: Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies. CONCLUSION: Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A/D HIV-1 recombinants, generated with each round of infection, are not replication-competent and do not survive in the multiple-cycle system. Ability of one HIV-1 isolate to outgrow the other leads to reduced co-infections, heterozygous virus production, and recombination frequencies

Sequence determinants of breakpoint location during HIV-1 intersubtype recombination

Retroviral recombination results from strand switching, during reverse transcription, between the two copies of genomic RNA present in the virus. We analysed recombination in part of the envelope gene, between HIV-1 subtype A and D strains. After a single infection cycle, breakpoints clustered in regions corresponding to the constant portions of Env. With some exceptions, a similar distribution was observed after multiple infection cycles, and among recombinant sequences in the HIV Sequence Database. We compared the experimental data with computer simulations made using a program that only allows recombination to occur whenever an identical base is present in the aligned parental RNAs. Experimental recombination was more frequent than expected on the basis of simulated recombination when, in a region spanning 40 nt from the 5′ border of a breakpoint, no more than two discordant bases between the parental RNAs were present. When these requirements were not fulfilled, breakpoints were distributed randomly along the RNA, closer to the distribution predicted by computer simulation. A significant preference for recombination was also observed for regions containing homopolymeric stretches. These results define, for the first time, local sequence determinants for recombination between divergent HIV-1 isolates

Characterization of a Subtype D Human Immunodeficiency Virus Type 1 Isolate That Was Obtained from an Untreated Individual and That Is Highly Resistant to Nonnucleoside Reverse Transcriptase Inhibitors

Human immunodeficiency virus type 1 (HIV-1) isolates derived from HIV-infected, treatment-naive Ugandan infants were propagated and tested for sensitivity to antiretroviral (ARV) drugs. Although most subtype A and D isolates displayed inhibition profiles similar to those of subtype B strains, a subtype D isolate identified as D14-UG displayed high-level resistance to nevirapine in peripheral blood mononuclear cell cultures (>2,000-fold) and in MT4 cell cultures (∼800-fold) but weaker resistance to delavirdine (∼13-fold) and efavirenz (∼8-fold) in MT4 cell cultures. To investigate the possible mechanism for this resistance to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs), the RT coding region in pol was sequenced and compared to the consensus RT sequence of NNRTI-resistant and NNRTI-sensitive subtype A, B, and D HIV-1 isolates. D14-UG did not contain the classic amino acid substitutions conferring NNRTI resistance (e.g., Y181C, K103N, and G190A) but did have some putative sites associated with drug resistance, I135L, T139V, and V245T. Wild-type and mutated protease-RT genes from D14-UG and an NNRTI-sensitive subtype D isolate from Uganda (D13-UG) were cloned into pNL4-3 to produce recombinant viruses and to determine the effects of the mutations on susceptibility to ARV drugs, specifically, NNRTIs. The results showed that I135L and/or V245T mutations can confer high-level resistance to nevirapine and delavirdine as well as low level cross-resistance to efavirenz. Finally, ex vivo fitness analyses suggested that NNRTI-resistant sites 135L and 245T in wild-type isolate D14-UG may reduce RT fitness but do not have an impact on the fitness of the primary HIV-1 isolate

Targets of Small Interfering RNA Restriction during Human Immunodeficiency Virus Type 1 Replication▿

Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5α did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5′ of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3′-to-5′ siRNA amplification and spreading. In contrast, HIV-1 RNA 3′ of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively “naked” cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5α, or other host restriction factors

Viral RNA silencing suppressors (RSS) : novel strategy of viruses to ablate the host RNA interference (RNAi) defense system

Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host&ndash;virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics