Diverse coronaviruses have been identified in bats from several continents but not from Africa. We identified group 1 and 2 coronaviruses in bats in Kenya, including SARS-related coronaviruses. The sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans.
The etiology of a large proportion of gastrointestinal illness is unknown. In this study, random Sanger sequencing and pyrosequencing approaches were used to analyze fecal specimens from a gastroenteritis outbreak of unknown etiology in a child care center. Multiple sequences with limited identity to known astroviruses were identified. Assembly of the sequences and subsequent reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends generated a complete genome of 6,586 nucleotides. Phylogenetic analysis demonstrated that this virus, named astrovirus VA1 (AstV-VA1), is highly divergent from all previously described astroviruses. Based on RT-PCR, specimens from multiple patients in this outbreak were unequivocally positive for Ast-VA1.
The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo.We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases.In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization.DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT.The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization.The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties.yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling.The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered.
The HIV integrase inhibitor, dolutegravir (DTG), in the absence of eliciting integrase (int) resistance, has been reported to select mutations in the virus 3'-polypurine tract (3'-PPT) adjacent to the 3'-LTR U3. An analog of DTG, cabotegravir (CAB), has a high genetic barrier to drug resistance and is used in formulations for treatment and long-acting pre-exposure prophylaxis. We examined whether mutations observed for DTG would emerge in vitro with CAB. HIV-1IIIB was cultured in paired experiments of continuous high (300 nM) CAB initiated 2 h or 24 h after infection. After eight months of CAB treatment, no int resistance was detected. Conversely, HIV RNA 3'-PPT mutants were detected within one month and were the majority virus by day 98. The appearance of 3'-PPT variants coincided with a rapid accumulation of HIV 1-LTR and 2-LTR circles. RNA amplification from the 3'-LTR TAR identified transcripts crossing 2-LTR circle junctions, which incorporated the adjacent U5 sequence identical to the 3'-PPT mutants. 3'-PPT variants were only identified in LTR circles and transcripts. Additionally, we found evidence of linear HIV and LTR circle recombination with human DNA at motifs homologous to 3'-PPT sequences. HIV persistence under CAB was associated with transcription and recombination of LTR circle sequences.
Pre-exposure prophylaxis (PrEP) with a weekly oral regimen of antiretroviral drugs could be a suitable preventative option for individuals who struggle with daily PrEP or prefer not to use long-acting injectables. We assessed in macaques the efficacy of weekly oral tenofovir alafenamide (TAF) at doses of 13.7 or 27.4 mg/kg. Macaques received weekly oral TAF for six weeks and were exposed twice-weekly to SHIV vaginally or rectally on day 3 and 6 after each dose. Median TFV-DP levels in PBMCs following the 13.7 mg/kg dose were 3110 and 1137 fmols/106 cells on day 3 and 6, respectively. With the 27.4 mg/kg dose, TFV-DP levels were increased (~2-fold) on day 3 and 6 (6095 and 3290 fmols/106 cells, respectively). Both TAF doses (13.7 and 27.4 mg/kg) conferred high efficacy (94.1% and 93.9%, respectively) against vaginal SHIV infection. Efficacy of the 27.4 mg/kg dose against rectal SHIV infection was 80.7%. We estimate that macaque doses of 13.7 and 27.4 mg/kg are equivalent to approximately 230 and 450 mg of TAF in humans, respectively. Our findings demonstrate the effectiveness of a weekly oral PrEP regimen and suggest that a clinically achievable oral TAF dose could be a promising option for non-daily PrEP.
The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins. Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT·nucleosome complexes. Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome. Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding. We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3. The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA. These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure. The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins. Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT·nucleosome complexes. Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome. Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding. We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3. The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA. These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure. Nhp6 is a DNA-binding protein that is encoded by two similar genes in Saccharomyces cerevisiae, NHP6A and NHP6B (1Kolodrubetz D. Burgum A. J. Biol. Chem. 1990; 265: 3234-3239Abstract Full Text PDF PubMed Google Scholar). Nhp6A and Nhp6B proteins (93 and 99 total residues, respectively) are 88% identical and functionally redundant (2Costigan C. Kolodrubetz D. Snyder M. Mol. Cell. Biol. 1994; 14: 2391-2403Crossref PubMed Scopus (108) Google Scholar, 3Kolodrubetz D. Kruppa M. Burgum A. Gene (Amst.). 2001; 272: 93-101Crossref PubMed Scopus (12) Google Scholar), so we refer here to both as Nhp6 protein. Nhp6 contains a single ∼70-residue high mobility group (HMG) 1The abbreviations used are: HMG, high mobility group; HMGB, high mobility group B; EMSA, electrophoretic mobility shift assay. box motif of the type found in the HMGB family (4Bustin M. Trends Biochem. Sci. 2001; 26: 152-153Abstract Full Text Full Text PDF PubMed Google Scholar). HMG proteins are abundant constituents of chromatin that fall into several families with distinct DNA-binding motifs (5Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (764) Google Scholar). HMG families are highly conserved and have been proposed to play many roles in chromatin function (5Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (764) Google Scholar, 6van Holde K.E. Chromatin. Springer-Verlag, New York1988Google Scholar, 7Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). Canonical HMGB proteins contain two tandem copies of the same HMG box motif, so Nhp6 is not a true representative of this family (4Bustin M. Trends Biochem. Sci. 2001; 26: 152-153Abstract Full Text Full Text PDF PubMed Google Scholar). However, no yeast protein has the canonical HMGB structure, so Nhp6 is the closest relative of this conserved family in yeast and is likely to provide clues about the functions of the HMG box motif both in HMGB proteins and in other contexts. Haploid yeast cells contain about 70,000 copies of Nhp6 (8Paull T.T. Carey M. Johnson R.C. Genes Dev. 1996; 10: 2769-2781Crossref PubMed Scopus (102) Google Scholar) for a total nuclear concentration of about 30 μm, roughly equivalent to nucleosomes. While abundant and conserved, Nhp6 is not essential for viability (2Costigan C. Kolodrubetz D. Snyder M. Mol. Cell. Biol. 1994; 14: 2391-2403Crossref PubMed Scopus (108) Google Scholar). However, yeast cells lacking Nhp6 display severe defects including slow growth, temperature sensitivity, altered regulation of transcription, and an inability to tolerate mutations in a variety of transcription and replication factors (2Costigan C. Kolodrubetz D. Snyder M. Mol. Cell. Biol. 1994; 14: 2391-2403Crossref PubMed Scopus (108) Google Scholar, 8Paull T.T. Carey M. Johnson R.C. Genes Dev. 1996; 10: 2769-2781Crossref PubMed Scopus (102) Google Scholar, 9Yu Y. Eriksson P. Stillman D.J. Mol. Cell. Biol. 2000; 20: 2350-2357Crossref PubMed Scopus (65) Google Scholar, 10Formosa T. Workman J.L. Protein Complexes That Modify Chromatin. Vol. 274. Springer-Verlag, Heidelberg, Germany2002: 171-201Google Scholar, 11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 12Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar). Nhp6 is therefore non-essential, but it is important for normal growth. Nhp6 supports the function of Spt16-Pob3, a heterodimer implicated in both DNA replication and regulation of transcription (10Formosa T. Workman J.L. Protein Complexes That Modify Chromatin. Vol. 274. Springer-Verlag, Heidelberg, Germany2002: 171-201Google Scholar, 11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 12Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar). Spt16 and Pob3 contain most of the sequences found in the components of the human FACT and frog DUF complexes (13Okuhara K. Ohta K. Seo H. Shioda M. Yamada T. Tanaka Y. Dohmae N. Seyama Y. Shibata T. Murofushi H. Curr. Biol. 1999; 9: 341-350Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 14Orphanides G. Wu W.H. Lane W.S. Hampsey M. Reinberg D. Nature. 1999; 400: 284-288Crossref PubMed Scopus (443) Google Scholar), but Pob3 lacks the HMG box motif found in the otherwise homologous FACT subunit SSRP1 (12Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar, 15Bruhn S.L. Pil P.M. Essigmann J.M. Housman D.E. Lippard S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2307-2311Crossref PubMed Scopus (236) Google Scholar, 16Wittmeyer J. Formosa T. Mol. Cell. Biol. 1997; 17: 4178-4190Crossref PubMed Google Scholar). Nhp6 appears to provide HMG box function for Spt16-Pob3 both in vivo and in vitro as cells lacking Nhp6 cannot tolerate mutations in SPT16 or POB3 (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 17Brewster N.K. Johnston G.C. Singer R.A. J. Biol. Chem. 1998; 273: 21972-21979Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and Spt16-Pob3 can only bind to and reorganize nucleosomes in vitro if Nhp6 is present (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar). Current data suggest that FACT family members, including Spt16-Pob3 with Nhp6 (SPN or yFACT), promote elongation by RNA and DNA polymerases by altering the structure of nucleosomes, making them less inhibitory to the passage of polymerases (14Orphanides G. Wu W.H. Lane W.S. Hampsey M. Reinberg D. Nature. 1999; 400: 284-288Crossref PubMed Scopus (443) Google Scholar, 18Orphanides G. LeRoy G. Chang C.-H. Luse D.S. Reinberg D. Cell. 1998; 92: 105-116Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Nhp6 is more abundant than Spt16-Pob3 (8Paull T.T. Carey M. Johnson R.C. Genes Dev. 1996; 10: 2769-2781Crossref PubMed Scopus (102) Google Scholar, 17Brewster N.K. Johnston G.C. Singer R.A. J. Biol. Chem. 1998; 273: 21972-21979Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Wittmeyer J. Joss L. Formosa T. Biochemistry. 1999; 38: 8961-8971Crossref PubMed Scopus (106) Google Scholar), does not form a stable complex with them (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 12Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar), and nhp6-Δ mutations cause phenotypes distinct from defects in SPT16 or POB3. These observations suggest that Nhp6 may function both within yFACT and in other pathways. HMG box proteins have been considered to act as “architectural” factors that bend DNA and induce juxtaposition of non-contiguous DNA sequences (5Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (764) Google Scholar, 7Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). Alternatively they have been proposed to reduce the overall stiffness of DNA by repeatedly binding to DNA and releasing it in a bent form, allowing the DNA to access a range of shapes more rapidly (8Paull T.T. Carey M. Johnson R.C. Genes Dev. 1996; 10: 2769-2781Crossref PubMed Scopus (102) Google Scholar, 20Ross E.D. Hardwidge P.R. Maher III, L.J. Mol. Cell. Biol. 2001; 21: 6598-6605Crossref PubMed Scopus (43) Google Scholar, 21Travers A.A. Ner S.S. Churchill M.E. Cell. 1994; 77: 167-169Abstract Full Text PDF PubMed Scopus (110) Google Scholar). This “shape chaperone” activity has been proposed to assist the formation of structures containing bent DNA, including nucleosomes (20Ross E.D. Hardwidge P.R. Maher III, L.J. Mol. Cell. Biol. 2001; 21: 6598-6605Crossref PubMed Scopus (43) Google Scholar, 21Travers A.A. Ner S.S. Churchill M.E. Cell. 1994; 77: 167-169Abstract Full Text PDF PubMed Scopus (110) Google Scholar). Here we report that the converse may also occur as Nhp6 binding appears to destabilize nucleosomes, promoting conversion to an alternative form. The conservation of the HMG box motif among FACT members and the importance of Nhp6 in yFACT activity suggest that this motif plays an important role in FACT function. The distinct architecture of yFACT allowed us to examine the function of the HMG box motif separately from Spt16-Pob3 to ask how this module contributes to yFACT-mediated reorganization of nucleosomes. Here we show that increasing concentrations of Nhp6 made progressive changes in nucleosome structure. At an Nhp6 concentration about 10-fold higher than the concentration that produces Nhp6·nucleosome complexes, nucleosomes underwent a change that led to enhanced DNase I sensitivity at some sites and the ability to bind Spt16-Pob3. These results suggest a general role for Nhp6 and other HMG proteins in destabilizing nucleosomes to promote formation of alternative chromatin structures. Nucleosomes—DNA molecules containing the 146-bp sea urchin 5 S rDNA nucleosome positioning sequence (22Simpson R.T. Stafford D.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 51-55Crossref PubMed Scopus (278) Google Scholar) were produced by PCR amplification using a template with the positioning sequence inserted between the EcoRI and XbaI sites in pBlueScript KS– (Stratagene, generously provided by J. Wittmeyer and B. Cairns). Primer sequences (available upon request) were designed that amplify the positioning sequence by PCR producing different flanking contexts as shown in Fig. 1. Products were end-labeled on one strand by digesting with an appropriate restriction endonuclease, treating with phosphatase, labeling with [γ-32P]ATP and polynucleotide kinase, and then digesting with a second endonuclease. Histone octamers were derived either from chicken erythrocytes (a gift from V. Ramakrishnan and V. Graziano, Ref. 23Graziano V. Gerchman S.E. Ramakrishnan V. J. Mol. Biol. 1988; 203: 997-1007Crossref PubMed Scopus (29) Google Scholar) or from bacteria expressing recombinant yeast histones (constructs generously provided by J. Wittmeyer and B. Cairns). Nucleosomes were assembled by slow dialysis from high salt solutions and purified by sucrose gradient centrifugation as described previously (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 24Luger K. Rechsteiner T.J. Richmond T.J. Methods Enzymol. 1999; 304: 3-19Crossref PubMed Scopus (574) Google Scholar). 2With modifications provided by J. Wittmeyer and B. Cairns, personal communication. Binding Reactions and DNase I Digestions—Spt16-Pob3 was purified as the intact heterodimer from yeast cells overexpressing both proteins as described previously (19Wittmeyer J. Joss L. Formosa T. Biochemistry. 1999; 38: 8961-8971Crossref PubMed Scopus (106) Google Scholar). Nhp6 was purified from BL21-Codon-Plus(DE3)-RIL Escherichia coli (Stratagene) expressing NHP6A from plasmid pRJ1228 (Ref. 25Paull T.T. Johnson R.C. J. Biol. Chem. 1995; 270: 8744-8754Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, a generous gift from R. Johnson). Cultures growing in rich medium at 37 °C were induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside for 4 h, harvested by centrifugation, suspended in lysis buffer (20 mm Tris-Cl, pH 8.0, 500 mm NaCl), and frozen. Cell suspensions were thawed, Nonidet P-40 was added to a final concentration of 0.1% (v/v), and the cells were lysed by sonication at 0 °C. Debris were removed by centrifugation at 17,000 × g for 30 min. Trichloroacetic acid was added to the cleared supernatant with constant mixing to 2% (w/v). The precipitate was removed by centrifugation at 39,000 × g for 30 min, and then trichloroacetic acid was added to the supernatant to 10% (w/v). The precipitate was harvested by centrifugation at 39,000 × g for 30 min, rinsed with acetone, dried, and then dissolved in S buffer (20 mm Tris-Cl, pH 7.5, 2 mm Na2EDTA, 1 mm 2-mercaptoethanol, 10% (w/v) glycerol, 300 mm NaCl). After dialyzing twice against 1 liter of fresh S buffer, the sample was loaded onto a 5-ml HiTrap SP column (Amersham Biosciences), which was washed with S buffer and then eluted with a 25-ml gradient from 300 mm NaCl to 1 m NaCl in S buffer. Fractions containing Nhp6 were dialyzed against S buffer, concentrated, and stored at –70 °C. Working stocks of proteins were made by diluting concentrated stocks in 20 mm Tris-Cl (pH 7.5), 100 mm NaCl, 1 mm Na2EDTA, 1 mm 2-mercaptoethanol, and 10% (w/v) glycerol. For binding reactions, proteins were mixed with 5–10 fmol of DNA or nucleosomes in a 10-μl volume containing final concentrations of 1 mm Na2EDTA, 20 mm HEPES (pH 7.6), 120 mm NaCl, 0.2 mm 2-mercaptoethanol, 0.9 mg/ml human serum albumin (Sigma), 12% (w/v) sucrose, and 2% (w/v) glycerol. After incubating for 10 min at 30 °C, products were separated by native PAGE for several hours at 100–200 V on 16- × 16- × 0.15-cm gels composed of 4% polyacrylamide (0.2% bisacrylamide), 5% (w/v) glycerol, 2 mm MgCl2, and 0.5× Tris borate/EDTA (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: B.23Google Scholar). Gels were dried, and labeled DNA was detected on a PhosphorImager (Storm 820, Amersham Biosciences). The amount of label in different regions of each lane was quantitated using ImageQuant software (Amersham Biosciences). In each case, the signal for a given form was determined and then normalized to the amount of signal in the entire lane to determine the percentage of the total DNA represented by the specific form. These values were then plotted to determine the point at which half of the maximum effect occurred as an estimate of the binding affinity. For DNase I digestions, 2 μl of a solution containing 100 ng of bovine pancreatic DNase I (Worthington), 60 mm MgCl2, 0.5 mg/ml human serum albumin, and 300 mm Tris-HCl (pH 7.5) was added to 10 μl of a binding reaction and incubated for 3 min at 30 °C. The reaction was stopped by adding 50 μl of a solution containing 6 μg of salmon testes DNA (Sigma), 3 mm Na2EDTA, 20 mm Tris-HCl (pH 7.5), and 100 mm NaCl at 0 °C. Samples were extracted with 150 μl of CHCl3 containing 125 volume of isoamyl alcohol, the aqueous phase was precipitated with 2.5 volumes of 95% ethanol, and the DNA was recovered by centrifugation. DNA was dissolved in 76% formamide, 16 mm Na2EDTA, and 0.04% each of bromphenol blue and xylene cyanol FF dyes. Samples were incubated for several minutes at 65 °C and then subjected to electrophoresis through 0.4-mm gels consisting of 8% polyacrylamide (0.4% bisacrylamide), 7 m urea, and 70% Tris borate/EDTA (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: B.23Google Scholar). Gels were fixed in 12% methanol and 10% acetic acid, dried, and then autoradiographed. Where indicated, the 12-μl DNase I digestions were mixed with 32 μg of salmon testes DNA, and then nucleosomes were recovered after native PAGE by extracting gel fragments overnight at 37 °C with 0.2% sodium dodecyl sulfate, 20 mm Tris-HCl (pH 7.5) followed by precipitation with ethanol. Nhp6 binds to DNA and to nucleosomes (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 25Paull T.T. Johnson R.C. J. Biol. Chem. 1995; 270: 8744-8754Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We have shown previously that Nhp6 promotes formation of complexes between Spt16-Pob3 and nucleosomes and that the enhanced sensitivity to DNase I associated with these yFACT· nucleosome complexes is partially induced at a subset of sites by Nhp6 alone (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar). This suggests that Nhp6 converts nucleosomes to an altered form in which the DNA is locally more accessible to DNase I or is a more suitable substrate for this nuclease and that unlike free nucleosomes this form is competent to bind Spt16-Pob3. DNA is dramatically bent by Nhp6 binding (27Allain F.H. Yen Y.M. Masse J.E. Schultze P. Dieckmann T. Johnson R.C. Feigon J. EMBO J. 1999; 18: 2563-2579Crossref PubMed Scopus (157) Google Scholar), and this could alter nucleosomes by disrupting local histone-DNA contacts. Nhp6 might bind to linker DNA and disturb contacts at the entry-exit points (linkers are defined here as the DNA extending beyond the 146-bp sea urchin 5 S rDNA nucleosome positioning sequence, Ref. 22Simpson R.T. Stafford D.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 51-55Crossref PubMed Scopus (278) Google Scholar). Alternatively Nhp6 could bind directly to the core DNA associated with the histone octamer, forcing this DNA into an aberrant shape incompatible with normal stable nucleosomal structure. These models make distinct predictions concerning the amount of Nhp6 required to alter nucleosomes and the effect of changing the size of the linkers. We therefore constructed nucleosomes with different linker configurations and tested them for the ability to bind Nhp6 or yFACT as well as for changes in sensitivity to DNase I. DNA and Nucleosomes Each Bind Multiple Monomers of Nhp6 —Nhp6 bound to either 149- or 237-bp linear double-stranded DNA fragments in an electrophoretic mobility shift assay (EMSA) with an apparent Kd of about 7 nm (Fig. 2A and Table I). As previously noted by the Johnson laboratory (8Paull T.T. Carey M. Johnson R.C. Genes Dev. 1996; 10: 2769-2781Crossref PubMed Scopus (102) Google Scholar, 25Paull T.T. Johnson R.C. J. Biol. Chem. 1995; 270: 8744-8754Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), multiple binding forms were observed as the concentration of Nhp6 was increased (Fig. 2A). These intermediates have been shown to reflect the binding of integral numbers of Nhp6 monomers (28Yen Y.M. Wong B. Johnson R.C. J. Biol. Chem. 1998; 273: 4424-4435Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), showing that DNA molecules of this size contain multiple binding sites for Nhp6 that can be accessed simultaneously. Nhp6 contacts 10–15 bp of DNA (27Allain F.H. Yen Y.M. Masse J.E. Schultze P. Dieckmann T. Johnson R.C. Feigon J. EMBO J. 1999; 18: 2563-2579Crossref PubMed Scopus (157) Google Scholar), so the 149-bp substrate shown in Fig. 2A should be able to bind 10–15 monomers of Nhp6. Very high concentrations of Nhp6 caused progressively slower migration of the DNA even after binding was saturated (Fig. 2A). This appears to be an artifact caused by Nhp6 molecules migrating in this region of the polyacrylamide gels independently of DNA (not shown; nucleosomes displayed the same effect, see Fig. 2, B and C). We estimated the affinity of Nhp6 for DNA or nucleosomes (Table I) by monitoring the loss of the unbound form, indicating binding by at least one monomer of Nhp6.Table IAffinities derived from EMSA experimentsnAffinity for Nhp6 (nm Nhp6)DNA (149 nts)107.2 ± 3.4DNA (237 nts)37.0 ± 2.6Summary137.1 ± 3.1NucleosomesYst, no linker1322 ± 18Yst, +linker430 ± 14Chk, no linker617 ± 5.5Chk, +linker322 ± 6.2Summary2622 ± 14Affinity for Nhp6 with SP (nm Nhp6)Chk ± linkers427 ± 13Yst ± linkers962 ± 46Summary1351 ± 42yFACT·Nuc formation (nm Nhp6)Yst, no linker9360 ± 160Yst, +linker3350 ± 120Chk, no linker8530 ± 120Chk, +linker3700 ± 100Summary23460 ± 180yFACT·Nuc formation (nm Spt16-Pob3)Chk, no linker88.7 ± 4.3Yst, no linker44.9 ± 1.4Chk, +linker1312 ± 3.2Yst, +linker48.5 ± 1.7Summary297.4 ± 3.7Nhp6 (yFACT·Nuc)/Nhp6 (Nuc binding)Chk520 ± 11Yst107.3 ± 2.7Summary1611 ± 8.4 Open table in a new tab We observed previously that Spt16-Pob3 and Nhp6 form a complex that can be observed in native PAGE under the low stringency conditions used for the EMSA (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar), but these proteins do not appear to form a stable complex under more physiological conditions (11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 12Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar). Addition of Spt16-Pob3 to the Nhp6-DNA binding reactions caused a slight shift in the migration pattern of the DNA (Fig. 2A, arrows) toward a lower Nhp6 concentration. Spt16-Pob3 therefore causes a small decrease in the effective concentration of Nhp6 for binding DNA, consistent with the interpretation that Spt16-Pob3 and Nhp6 do not form a specific, independent, active complex analogous to human FACT. A comparable ladder of binding intermediates was observed with nucleosomes (Fig. 2, B and C), suggesting that nucleosomes also have multiple binding sites for Nhp6. Due to the smaller proportional change in mass and differences in migration properties caused by different linker configurations, distinct ladders were not always observed, making it difficult in some cases to clearly discriminate the unbound form. This contributes to a high variance in measurements of the affinity of Nhp6 for nucleosomes, but we observed an average affinity with different types of nucleosomes of about 22 nm for Nhp6 (Table I). Nucleosomes and free DNA therefore appear to have a similar number of binding sites for Nhp6, and the affinity of the first binding event is about 3-fold lower for nucleosomes than for free DNA. Linkers Do Not Promote Nhp6 or yFACT Binding—If linkers provide a more efficient binding site for Nhp6 than DNA within the nucleosomal core, then nucleosomes with linkers should have higher affinity for Nhp6 than nucleosomes without linkers. However, the presence or absence of linkers on nucleosomes made little, if any, change in their affinity for Nhp6 (Table I). If Nhp6 promotes yFACT·nucleosome complex formation by affecting nucleosomes specifically at entry/exit points, then linkers might promote complex formation without altering the overall affinity for Nhp6. We therefore compared the ability of Nhp6 to support the formation of yFACT complexes using nucleosomes with and without linkers. As shown in Table I, the presence of linkers did not enhance the formation of yFACT complexes. For example, nucleosomes formed with chicken histones formed half of the maximal level of complexes at 530 nm Nhp6 without linkers and 700 nm Nhp6 with linkers. Linkers are therefore either neutral, or they slightly inhibit the ability of nucleosomes either to bind Nhp6 or to form yFACT complexes. Formation of yFACT·Nucleosome Complexes Requires Multiple Nhp6 Molecules—If binding of a single monomer of Nhp6 to a nucleosome is adequate to convert the nucleosome to a state competent for recruiting Spt16-Pob3, then formation of yFACT·nucleosome complexes should occur at the same concentration of Nhp6 as simple binding. Instead half-maximal formation of yFACT complexes required about 10-fold more Nhp6 than that needed for simple binding (Table I). Fig. 2 (B–D) shows that yFACT·nucleosome complexes only began to accumulate after the concentration of Nhp6 reached a level sufficient to drive essentially all nucleosomes into complexes with multiple Nhp6 molecules. As with free DNA, addition of Spt16-Pob3 caused a decrease in the effective concentration of Nhp6, so the apparent affinity of Nhp6 for nucleosomes in the presence of Spt16-Pob3 was about 50 nm Nhp6 (Table I). In contrast, the overall average for half-maximal formation of yFACT·nucleosome complexes in the same experiments was 460 nm Nhp6 (Table I). The average ratio of Nhp6 required for yFACT complex formation to simple binding in 16 independent experiments was 11 (Table I). It therefore appears that binding a single Nhp6 molecule is not adequate to support the formation of a yFACT·nucleosome complex. Histone octamers purified from chicken erythrocytes may retain some of the covalent modifications that occur in vivo (6van Holde K.E. Chromatin. Springer-Verlag, New York1988Google Scholar), and chicken histones do not have the same sequences as yeast histones (∼70% identity for H2A-H2B and 90% identity for H3-H4, Ref. 29Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). Mutations in yFACT subunits cause synthetic effects when combined with mutations in either histones or histone-modifying enzymes (9Yu Y. Eriksson P. Stillman D.J. Mol. Cell. Biol. 2000; 20: 2350-2357Crossref PubMed Scopus (65) Google Scholar, 11Formosa T. Eriksson P. Wittmeyer J. Ginn J. Yu Y. Stillman D.J. EMBO J. 2001; 20: 3506-3517Crossref PubMed Scopus (207) Google Scholar, 30Formosa T. Ruone S. Adams M.D. Olsen A.E. Eriksson P. Yu Y. Rhoades A.R. Kaufman P.D. Stillman D.J. Genetics. 2002; 162: 1557-1571Crossref PubMed Google Scholar), so Nhp6 and yFACT activities could be affected by either histone modifications or altered histone sequences. As shown in Table I, nucleosomes assembled using endogenous chicken histone octamers required somewhat higher levels of Nhp6 to form yFACT· nucleosome complexes than nucleosomes assembled with unmodified recombinant yeast histone octamers. For example, nucleosomes with linkers formed yFACT·nucleosome complexes with a half-maximum of 350 nm Nhp6 when yeast histones were used and 700 nm with chicken histones. This effect was small but consistent; an example of a single experiment in which the same DNA was assembled into yeast or chicken nucleosomes for direct comparison is shown in Fig. 3A. Simple binding of Nhp6 was similar for yeast and chicken nucleosomes, but yeast nucleosomes required 7-fold more Nhp6 to produce yFACT complexes, whereas chicken nucleosomes required 20-fold more (Table I). This suggests that increasing the concentration of Nhp6 causes progressive changes in nucleosomes, and the threshold level required to allow binding of Spt16-Pob3 is partly dependent on either the sequence of the histones or the presence of modifications. Spt16-Pob3 Binds with High Affinity—Spt16-Pob3 formed yFACT·nucleosome complexes in the presence of saturating amounts of Nhp6 with an overall apparent Kd of about 7 nm (Fig. 2D and Table I). Either the presence of linkers or the use of chicken histones caused a requirement for somewhat higher concentrations of Spt16-Pob3 relative to yeast nucleosomes without linkers (Table I and Fig. 3, B and C). The effects were small but reproducible within individual direct comparisons (Fig. 3B). We also noted that a smaller p
Abstract Studies in SIV-infected macaques show that the virus reservoir is particularly refractory to conventional suppressive antiretroviral therapy (ART). We posit that optimized ART regimens designed to have robust penetration in tissue reservoirs and long-lasting antiviral activity may be advantageous for HIV or SIV remission. Here we treat macaques infected with RT-SHIV with oral emtricitabine/tenofovir alafenamide and long-acting cabotegravir/rilpivirine without (n = 4) or with (n = 4) the immune activator vesatolimod after the initial onset of viremia. We document full suppression in all animals during treatment (4-12 months) and no virus rebound after treatment discontinuation (1.5-2 years of follow up) despite CD8 + T cell depletion. We show efficient multidrug penetration in virus reservoirs and persisting rilpivirine in plasma for 2 years after the last dose. Our results document a type of virus remission that is achieved through early treatment initiation and provision of ultra long-lasting antiviral activity that persists after treatment cessation.
Defects in SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae Cause Dependence on the Hir/Hpc Pathway: Polymerase Passage May Degrade Chromatin Structure Tim Formosa *¥ , Susan Ruone * , Melissa D. Adams †1 , Aileen E. Olsen ‡ , Peter Eriksson ‡2 , Yaxin Yu ‡ , Alison R. Rhoades * , Paul D. Kaufman † , and David J. Stillman ‡ University of Utah School of Medicine, Departments of * Biochemistry and ‡ Pathology, Salt Lake City, 84132, † Lawrence Berkeley National Laboratory and University of California, Berkeley, Department of Molecular and Cell Biology, Berkeley California 94720 Current address: Department of Biology, University of North Carolina, Chapel Hill, NC 27599 Current address: National Institutes of Health, Bethesda, MD 20892
Despite the immunologic protection associated with routine vaccination protocols, Canine distemper virus (CDV) remains an important pathogen of dogs. Antemortem diagnosis of systemic CDV infection may be made by reverse transcription polymerase chain reaction (RT-PCR) and/or immunohistochemical testing for CDV antigen; central nervous system infection often requires postmortem confirmation via histopathology and immunohistochemistry. An 8-month-old intact male French Bulldog previously vaccinated for CDV presented with multifocal neurologic signs. Based on clinical and postmortem findings, the dog's disease was categorized as a meningoencephalitis of unknown etiology. Broadly reactive, pan-paramyxovirus RT-PCR using consensus-degenerate hybrid oligonucleotide primers, combined with sequence analysis, identified CDV amplicons in the dog's brain. Immunohistochemistry confirmed the presence of CDV antigens, and a specific CDV RT-PCR based on the phosphoprotein gene identified a wild-type versus vaccinal virus strain. This case illustrates the utility of broadly reactive PCR and sequence analysis for the identification of pathogens in diseases with unknown etiology.
Daily oral pre- or post-exposure prophylaxis (PrEP or PEP) is highly effective in preventing HIV infection. However, many people find it challenging to adhere to a daily oral regimen. Chemoprophylaxis with single oral doses of antiretroviral drugs taken before or after sex may better adapt to changing or unanticipated sexual practices and be a desirable alternative to daily PrEP or PEP. We investigated willingness to use a single oral pill before or after sex among men who have sex with men (MSM) and assessed the biological efficacy of a potent antiretroviral combination containing elvitegravir (EVG), emtricitabine (FTC), and tenofovir alafenamide (TAF).
