Schematic Representation of the Genomic Organization of Chimeric HIV/MLV Proviruses

<p>Portions originated from the HIV genome are shown in white, while those from the MLV genome are in gray. The junction between HIV-1 RT and MLV IN within HIV-mIN was created by direct joining of DNA sequences encoding the C-terminus of HIV RT to the N-terminus of MLV IN. Part of the 3′ end of the HIV-1 IN encoding sequence is retained in the construct of MHIV-mIN to preserve the overlapping Vif sequence and <i>cis</i>-acting elements such as cPPT and splice acceptor(s). However, no part of HIV IN should be expressed in the chimeric virus because of the presence of two stop codons following the sequence encoding MLV IN (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#s4" target="_blank">Materials and Methods</a>). The molecular clone encoding MHIV-mIN/m<i>att</i> has the MLV <i>att</i> sites in 5′ U5 and 3′ U3. After reverse transcription, both ends of U5 and U3 will have the MLV <i>att</i> sites. MHIV-mMA12CA has been previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#ppat-0010018-b040" target="_blank">40</a>]. MHIV-mMA12CA/mIN is similar except it contains MLV IN in addition to the MLV Gag region. A molecular clone of MHIV-mMA12CA/mIN was created by putting the DNA sequence encompassing the MLV IN encoding sequence of the MHIV-mIN with a Vpr mutation into the infectious provirus pMHIV-mMA12CA. HIVΔNLS contains MLV MA instead of HIV MA, MLV IN instead of HIV IN and mutations in the cPPT and in Vpr.</p

Integration Sites of MHIV-mIN

<p>A schematic illustration of the unintegrated viral DNA is shown with the detailed structures of both ends of LTR (top). The two terminal base pairs at each end of the linear DNA precursor, which are removed in the integration process, as shown in highlight. Two other DNA sequences confirmed the removal of dinucleotides from one end of viral DNA (unpublished data). DNA sequences flanking integrated proviral DNA are shown (bottom). Junction sequences between the integrated MHIV-mIN genome and human genomic DNA at each end of the provirus were obtained by nested PCR based on the sequence of integration sites that were mapped with human genome sequences as described in the Materials and Methods. The 4-bp sequence duplications that flank the integrated provirus are shown in boxes.</p

IN Does Not Determine the Infectivity in Non-Dividing Cells

<p>Aphidicolin-treated HeLa cells were infected with increasing amount of luciferase-encoding viruses described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#ppat-0010018-g001" target="_blank">Figure 1</a>. Culture supernatants of transfected cells were used as the inoculum. Virus infectivity was judged by measuring luciferase titers of infected cell lysates 2 d after infection. RLU; relative light units. White circles indicate cells without aphidicolin. Filled circles indicate cells with aphidicolin (2 μg per ml). This is a representative experiment that was done at least three times for each virus.</p

Western Blot Analysis of Purified Virus Particles of MHIV-mIN Together with HIV-1 and MLV

<p>Polyprotein processing was tested for HIV-1 RT, HIV-1 IN and MLV IN. Because of low protein production by MHIV-mIN, 10-times more virions were loaded for MHIV-mIN than for HIV-1 and MLV in this experiment.</p

MHIV-mIN Is Infectious

<div><p>(A) Single-cycle infectivity of MHIV-mIN. VSV-G-pseudotyped HIV and MHIV-mIN were made by transfection of 293T cells with plasmid DNA. Infectivity was measured with the MAGI assay by counting β-galactosidase positive cells 2 d post-infection. Virus titers were normalized by the amount of p24 (μg). Infections were performed in triplicate. Mean values are shown here with standard derivation. The background in the assay is about 10 blue cells.</p><p>(B) Copy numbers of late products of reverse transcription were measured by using real-time quantitative PCR. Viral cDNA numbers were normalized by p24. Infections were performed in triplicate. This is a representative experiment of three independent trials.</p><p>(C) Infectivity of MHIV-mIN and MHIV-mIN/m<i>att</i> was compared in the MAGI assay. VSV-G-pseudotyped viruses were prepared and concentrated at 100-fold by ultracentrifugation. Infections of MAGI cells were also performed in the presence or absence of reverse transcriptase inhibitors (50 μM 3TC and AZT). Formation of blue cells in this assay must result from retrovirus infection since addition of reverse transcription inhibitors (shown as black bars) eliminated most of positive cells.</p><p>(D) Comparison of infectivity between wild-type HIV-1 and MHIV-mIN as judged by the ability to make puromycin-resistant clones. Infectivity was measured by counting puromycin-resistant colonies 14 d after infection. To facilitate stable transduction of HIV genomes, a mutation was introduced into the <i>vpr</i> gene in these proviruses because expression of Vpr would preclude formation of colonies due to its cytotoxicity [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#ppat-0010018-b069" target="_blank">69</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#ppat-0010018-b077" target="_blank">77</a>]</p></div

HIV Lacking NLSs Infects Non-Dividing Cells

<p>Single-cycle infectivity assay of HIV-ΔNLS together with HIV, MLV, and MHIV-mMA12CA. Both HIV-ΔNLS and MHIV-mMA12CA were concentrated by ultracentrifugation for infections. For details, see the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010018#ppat-0010018-g005" target="_blank">Figure 5</a>. White circles indicate cells without aphidicolin. Filled circles indicate cells with aphidicolin (2 μg per ml). This is a representative experiment that was done at least 3 times for each virus.</p

Nuclear Entry of MHIV-mIN

<div><p>(A) Subcellular localization of viral DNA. Infected cells were fractionated to cytoplasmic (C) and nuclear (N) fractions. Viral DNA was extracted and subject to real-time PCR to measure late products of reverse transcription. These data represent one of two independent experiments. Control viruses, with the presence of reverse transcription inhibitors or without VSV-G protein, were also used in the experiments to monitor retrovirus-dependent DNA synthesis, and showed that contamination of plasmid DNA used for transfection is less than 1% of the total DNA.</p><p>(B) Western blot analysis of total cell lysates (To), cytoplasmic extract (Cy) and nuclear lysates (Nu). Contamination of cytoplasmic extract and the presence of intact cells in nuclear fractions were tested by checking for the presence of a cytoplasmic protein, LDH-I, in each fraction (upper lanes). Five-fold dilutions of the cytoplasmic extract (5-, 25-, and 125-fold dilutions, lanes 4, 3, and 2, respectively) were made to assess the degree of contamination of the nuclear fraction. Presence of proteins was confirmed by antibody against a nuclear pore complex protein (mAb414; lower lane).</p><p>(C) Nuclear import was monitored by measuring late reverse transcription products and 2-LTR circles. The ratio of total viral DNA and 2-LTR circles was obtained by dividing the copy number of late RT products by the copy number of 2-LTR circle. The parental wild-type strain of HIV-1 (shown here as wt) was compared with the chimeric virus MHIV-mIN (shown as mIN). Control infections with reverse transcription inhibitors (AZT and 3TC: 50 μM each) yielded viral copy numbers that are less than 10% of copy numbers of the samples without reverse transcription inhibitors, indicating that contamination of plasmid DNA used to produce virus stocks does not affect the final results. Two independent experiments gave substantially identical data.</p></div

Cyanide-Bridged Fe(III)−Mn(III) Chain with Metamagnetic Properties and Significant Magnetic Anisotropy

The assembling of [Mn(5-MeOsalen)(H2O)]+ and [(Tp)Fe(CN)3]- affords the one-dimensional zigzag chain [(Tp)Fe(CN)3Mn(5-MeOsalen)·2CH3OH]n [1; Tp- = hydrotris(pyrazolyl)borate and 5-MeOsalen2- = N,N‘-ethylenebis(5-methoxysalicylideneiminate)]. The corroborated experimental and ab initio data indicate ferromagnetic Fe(III)−Mn(III) couplings and D < 0 anisotropy on Mn(III). The field-induced metamagnetic behavior is due to interchain effects

Cyanide-Bridged Fe(III)−Mn(III) Chain with Metamagnetic Properties and Significant Magnetic Anisotropy

The assembling of [Mn(5-MeOsalen)(H2O)]+ and [(Tp)Fe(CN)3]- affords the one-dimensional zigzag chain [(Tp)Fe(CN)3Mn(5-MeOsalen)·2CH3OH]n [1; Tp- = hydrotris(pyrazolyl)borate and 5-MeOsalen2- = N,N‘-ethylenebis(5-methoxysalicylideneiminate)]. The corroborated experimental and ab initio data indicate ferromagnetic Fe(III)−Mn(III) couplings and D < 0 anisotropy on Mn(III). The field-induced metamagnetic behavior is due to interchain effects

Coordination Assemblies of [Mn₄] Single-Molecule Magnets Linked by Photochromic Ligands: Photochemical Control of the Magnetic Properties

To achieve reversible photoswitching of the magnetic properties of single-molecule magnets (SMMs), coordination assemblies of a mixed valence tetranuclear [MnII2MnIII2] complex linked by open- and closed-ring isomers of a photochromic diarylethene ligand were synthesized: [Mn4(hmp)6(dae-o)2(ClO4)2]·6H2O (open-ring form; 1o) and [Mn4(hmp)6(dae-c)2(H2O)2](ClO4)2·CH3CN·4H2O (closed-ring form; 1c), where Hhmp is 2-hydroxymethylpyridine, and H2dae-o and H2dae-c are open- and closed-ring isomers of 1,2-bis(5-caxboxyl-2-methyl-3-thienyl)perfluorocyclopentene (H2dae), respectively. From X-ray crystallographic analyses, 1o and 1c have one-dimensional (1-D) chain structures with a repeating unit of [−{Mn4}−(dae)−], in which the [Mn4] units are bridged by the diarylethene ligands coordinated to the MnII sites via carboxylate groups. Magnetic measurements involving direct-current (dc) and alternating-current (ac) techniques showed that each [Mn4] cluster in the 1-D chains had an ST = 9 ground state due to two kinds of intracluster ferromagnetic exchange interactions and behaved as SMMs. Both 1o and 1c underwent photochromic reactions in the crystalline phase, and their colors reversibly changed between reddish-brown and blue upon irradiation with ultraviolet (UV) and visible light. For 1o, no significant change in the magnetic properties was observed after the photocyclization of the dae-o moiety by UV irradiation. In contrast, the photocycloreversion of the dae-c moiety by visible irradiation enhanced inter-[Mn4] unit interactions in 1c, which was triggered by the geometrical change in the diarylethene moiety during the photoreaction and the subsequent change in the inter-[Mn4] arrangement between the neighboring 1-D chains. This work illustrates the usefulness of photochromic molecules for photoswitching the magnetic behavior of complexes, even superparamagnetic systems, with SMM units