GOLGA8 increases bulk antisense oligonucleotide uptake and activity in mammalian cells.

Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically.

Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA.

CRISPR-based gene editing in mammalian cells is a powerful research tool which has demonstrated efficient site-specific gene modifications and is showing promise as a therapeutic for patients with genetic diseases. Multiple different CRISPR systems have been identified, each with its own target DNA recognition sequence, expanding the editable mammalian genome. It is also now appreciated that chemically modified nucleic acids can substitute for unmodified nucleotides in guide RNAs, providing protection from exonuclease degradation and improving gene editing efficiency.

DNA replication acts as an error correction mechanism to maintain centromere identity by restricting CENP-A to centromeres.

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms.

Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear transactive response DNA-binding protein 43 (TDP-43). Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA.

TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC.

The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC.

Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells.

CRISPR-based gene editing is a powerful technology for engineering mammalian genomes. It holds the potential as a therapeutic, although much-needed in vivo delivery systems have yet to be established. Here, using the Cpf1-crRNA (CRISPR RNA) crystal structure as a guide, we synthesized a series of systematically truncated and chemically modified crRNAs, and identify positions that are amenable to modification while retaining gene-editing activity.

CENP-A Is Dispensable for Mitotic Centromere Function after Initial Centromere/Kinetochore Assembly.

Human centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive alphoid DNA sequences. By inducing rapid, complete degradation of endogenous CENP-A, we now demonstrate that once the first steps of centromere assembly have been completed in G1/S, continued CENP-A binding is not required for maintaining kinetochore attachment to centromeres or for centromere function in the next mitosis. Degradation of CENP-A prior to kinetochore assembly is found to block deposition of CENP-C and CENP-N, but not CENP-T, thereby producing defective kinetochores and failure of chromosome segregation.

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion.

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain.

Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA.

DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function.

Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence-specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A's amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly.

Generation of an HIV resistant T-cell line by targeted "stacking" of restriction factors.

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process.

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration.

HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway.

Consistent inhibition of HIV-1 replication in CD4+ T cells by acyclovir without detection of human herpesviruses.

Acyclovir, a nucleoside analog, is thought to be specific for the human herpesviruses because it requires a virally encoded enzyme to phosphorylate it to acyclovir monophosphate. Recently, acyclovir triphosphate was shown to be a direct inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Here, we showed that acyclovir is an inhibitor of HIV-1 replication in CD4(+) T cells from cord blood that have undetectable levels of the eight human herpesviruses.

Short communication: dynamic constraints on the second phase compartment of HIV-infected cells.

The cells responsible for the second phase decay of HIV-1 viremia following the initiation of antiretroviral therapy have yet to be identified. A dynamic model that considers where drugs act in the virus life cycle places constraints on candidate cell types. In this regard, the rapid drop in viremia in patients starting regimens containing the integrase inhibitor raltegravir is of particular interest.

Local sequence targeting in the AID/APOBEC family differentially impacts retroviral restriction and antibody diversification.

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G).

Effect of acyclovir on HIV-1 set point among herpes simplex virus type 2-seropositive persons during early HIV-1 infection.

We evaluated whether acyclovir suppression during human immunodeficiency virus type 1 (HIV-1) acquisition reduces HIV-1 set point, increases CD4 cell counts, and selects reverse-transcriptase mutations among 76 HIV-1 seroconverters identified in a placebo-controlled trial of twice-daily acyclovir (400 mg) for the prevention of HIV acquisition in herpes simplex virus type 2 (HSV-2)-seropositive persons (HIV Prevention Trials Network study 039). We found no significant difference in plasma HIV-1 RNA levels (P =.30) or CD4 cell counts (P =.

Sensitivity of V75I HIV-1 reverse transcriptase mutant selected in vitro by acyclovir to anti-HIV drugs.

Trials of acyclovir for herpes simplex virus 2 infection in herpes simplex virus 2/HIV-1 coinfected patients not on antiretroviral therapy demonstrated a decrease in herpes simplex virus 2 and HIV-1 replication. Recent studies indicated that acyclovir has direct anti-HIV-1 activity and can select for the HIV-1 V75I reverse transcriptase variant in vitro. We show that the V75I variant has decreased sensitivity to some nucleoside analogs but an increased sensitivity to zidovudine, results that may guide selection of highly active antiretroviral therapy regimens in patients harboring this variant.

New approaches for quantitating the inhibition of HIV-1 replication by antiviral drugs in vitro and in vivo.

Purpose Of Review: With highly active antiretroviral therapy, HIV-1 infection has become a manageable lifelong disease. Developing optimal treatment regimens requires understanding how to best measure anti-HIV activity in vitro and how drug dose-response curves generated in vitro correlate with in-vivo efficacy.

Recent Findings: Several recent studies have indicated that conventional multiround infectivity assays are inferior to single cycle assays at both low and high levels of inhibition.

The antiherpetic drug acyclovir inhibits HIV replication and selects the V75I reverse transcriptase multidrug resistance mutation.

The antiviral drug acyclovir is a guanosine nucleoside analog that potently inhibits herpes simplex virus (HSV) replication. Acyclovir treatment in patients coinfected with HSV and human immunodeficiency virus (HIV) has been observed to alter disease course and decrease HIV viral load, a finding that has been attributed to indirect effects of HSV suppression on HIV replication. Based on this hypothesis, several clinical studies have recently investigated the use of acyclovir for treatment of patients coinfected with HSV and HIV or for prophylaxis against HIV transmission.

Dose-response curve slope sets class-specific limits on inhibitory potential of anti-HIV drugs.

Highly active antiretroviral therapy (HAART) can control HIV-1 replication, but suboptimal treatment allows for the evolution of resistance and rebound viremia. A comparative measure of antiviral activity under clinically relevant conditions would guide drug development and the selection of regimens that maximally suppress replication. Here we show that current measures of antiviral activity, including IC(50) and inhibitory quotient, neglect a key dimension, the dose-response curve slope.

The anti-HIV activity of entecavir: a multicentre evaluation of lamivudine-experienced and lamivudine-naive patients.

Background: Entecavir, an antiviral with potent anti-hepatitis B virus activity, was recently shown to have anti-HIV activity in three patients and the ability to select for the lamivudine-resistant HIV polymerase mutation M184V in a patient with prior antiretroviral therapy.

Objectives: To further characterize entecavir's anti-HIV activity and identify risk factors for selection of the M184V.

Design: Retrospective cohort study.

The HBV drug entecavir - effects on HIV-1 replication and resistance.

Entecavir, a drug approved by the Food and Drug Administration for the treatment of chronic hepatitis B virus (HBV) infection, is not believed to inhibit replication of human immunodeficiency virus type 1 (HIV-1) at clinically relevant doses. We observed that entecavir led to a consistent 1-log(10) decrease in HIV-1 RNA in three persons with HIV-1 and HBV coinfection, and we obtained supportive in vitro evidence that entecavir is a potent partial inhibitor of HIV-1 replication. Detailed analysis showed that in one of these patients, entecavir monotherapy led to an accumulation of HIV-1 variants with the lamivudine-resistant mutation, M184V.

Multiple targeting modules on peroxisomal proteins are not redundant: discrete functions of targeting signals within Pmp47 and Pex8p.

Several peroxisomal proteins have two nonoverlapping targeting signals. These signals have been termed "redundant" because targeting can still occur with only one signal. We now report that separate targeting motifs within both Pmp47 and Pex8 provide complementary function.