The membrane associated accessory protein is an adeno-associated viral egress factor

AbstractAdeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

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The membrane associated accessory protein is an adeno-associated viral egress factor

10.1101/2021.06.17.448857 ◽

2021 ◽

Author(s):

Zac Elmore ◽

L Patrick Havlik ◽

Daniel K Oh ◽

Heather A Vincent ◽

Aravind Asokan

Keyword(s):

Extracellular Vesicle ◽

Lytic Infection ◽

Open Reading Frame ◽

Accessory Protein ◽

Start Site ◽

Wild Type ◽

Reading Frame ◽

Extracellular Secretion ◽

Viral Egress

Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some recombinant AAV serotypes are secreted in a pre-lytic manner as extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a (+1) frameshifted open reading frame (ORF) in the AAV capsid (cap) gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with recombinant MAAP restored extracellular secretion of multiple AAV/MAAPΔ serotypes. MAAP is sorted into recycling Rab11+ vesicles and strongly associates with EV markers upon fractionation. In addition to characterizing a novel viral egress factor, these studies highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

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The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽

10.1093/genetics/155.3.1105 ◽

2000 ◽

Vol 155 (3) ◽

pp. 1105-1117 ◽

Cited By ~ 4

Author(s):

W John Haynes ◽

Kit-Yin Ling ◽

Robin R Preston ◽

Yoshiro Saimi ◽

Ching Kung

Keyword(s):

Genomic Library ◽

Open Reading Frame ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Rt Pcr ◽

Reading Frame ◽

Close Proximity ◽

In The Wild ◽

Dna Fragment ◽

Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

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Regulation of the nfsA Gene in Escherichia coli by SoxS

Journal of Bacteriology ◽

10.1128/jb.184.1.51-58.2002 ◽

2002 ◽

Vol 184 (1) ◽

pp. 51-58 ◽

Cited By ~ 27

Author(s):

E. Suzanne Paterson ◽

Sherri E. Boucher ◽

I. B. Lambert

Keyword(s):

Escherichia Coli ◽

Promoter Sequence ◽

Open Reading Frame ◽

Base Sequence ◽

Start Site ◽

Transcription Start ◽

Band Shift ◽

Reading Frame ◽

Shift Analysis ◽

Small Open Reading Frame

ABSTRACT In Escherichia coli, the response to oxidative stress due to elevated levels of superoxide is mediated, in part, by the soxRS regulon. One member of the soxRS regulon, nfsA, encodes the major oxygen-insensitive nitroreductase in Escherichia coli which catalyzes the reduction of nitroaromatic and nitroheterocyclic compounds by NADPH. In this study we investigate the regulation of nfsA in response to the superoxide generating compound paraquat. The transcription start site (TSS) of nfsA was located upstream of the ybjC gene, a small open reading frame of unknown function located directly upstream of nfsA, suggesting that these two genes form an operon. The activity of the promoter associated with this TSS was confirmed with lacZ fusions and was shown to be inducible by paraquat. Footprinting and band shift analysis showed that purified His-tagged SoxS protein binds to a 20-base sequence 10 bases upstream of the −35 promoter sequence in the forward orientation, suggesting that the ybjC-nfsA promoter is a class I SoxS-dependent promoter.

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CZK3, a MAP Kinase Kinase Kinase Homolog in Cercospora zeae-maydis, Regulates Cercosporin Biosynthesis, Fungal Development, and Pathogenesis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.9.760 ◽

2003 ◽

Vol 16 (9) ◽

pp. 760-768 ◽

Cited By ~ 41

Author(s):

Won-Bo Shim ◽

Larry D. Dunkle

Keyword(s):

Map Kinase ◽

Map Kinases ◽

Gray Leaf Spot ◽

Open Reading Frame ◽

Wild Type ◽

Toxic Activity ◽

Reading Frame ◽

Cercospora Zeae Maydis ◽

Map Kinase Kinase Kinase ◽

Map Kinase Kinase

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

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Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2593-2608.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2593-2608 ◽

Cited By ~ 1

Author(s):

D X Tishkoff ◽

A W Johnson ◽

R D Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Open Reading Frame ◽

Strand Exchange ◽

Wild Type ◽

Homologous Pairing ◽

Gene Encoding ◽

Reading Frame ◽

Cloned Gene ◽

Diploid Cells ◽

Exchange Protein

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

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The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1506 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1506-1516 ◽

Cited By ~ 49

Author(s):

Ying Cui ◽

Jonathan D. Dinman ◽

Terri Goss Kinzy ◽

Stuart W. Peltz

Keyword(s):

Site Selection ◽

Start Site ◽

Wild Type ◽

Virus Propagation ◽

Ribosomal Frameshifting ◽

Reading Frame ◽

Translational Accuracy ◽

Its Gene ◽

Killer Virus

ABSTRACT Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene.mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

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Cloning and Characterization of a Prolinase Gene (pepR) from Lactobacillus rhamnosus

Applied and Environmental Microbiology ◽

10.1128/aem.64.5.1831-1836.1998 ◽

1998 ◽

Vol 64 (5) ◽

pp. 1831-1836 ◽

Cited By ~ 21

Author(s):

Pekka Varmanen ◽

Terhi Rantanen ◽

Airi Palva ◽

Soile Tynkkynen

Keyword(s):

Lactobacillus Rhamnosus ◽

Gene Replacement ◽

Lactobacillus Helveticus ◽

Open Reading Frame ◽

Transcriptional Unit ◽

Wild Type ◽

Reading Frame ◽

Gene Expressing ◽

Produce Acid

ABSTRACT A peptidase gene expressingl-proline-β-naphthylamide-hydrolyzing activity was cloned from a gene library of Lactobacillus rhamnosus 1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kbSacI fragment. A sequence analysis of the SacI fragment revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity with the PepR protein from Lactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) following pepR; this open reading frame was 459 bp long. Northern (RNA) and primer extension analyses indicated that pepR is expressed both as a monocistronic transcriptional unit and as a dicistronic transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain of L. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The clonedpepR expressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substrates l-leucine-β-naphthylamide andl-phenylalanine-β-naphthylamide were hydrolyzed by the PepR of L. rhamnosus.

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Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2374 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2374-2382 ◽

Cited By ~ 28

Author(s):

M Bernstein ◽

W Hoffmann ◽

G Ammerer ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Gene Fusion ◽

Protein Assembly ◽

Open Reading Frame ◽

Cell Fractionation ◽

Multicopy Plasmid ◽

Wild Type ◽

Cytosol Fraction ◽

Reading Frame

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event.

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Deletion of Open Reading Frame UL26 from the Human Cytomegalovirus Genome Results in Reduced Viral Growth, Which Involves Impaired Stability of Viral Particles

Journal of Virology ◽

10.1128/jvi.02585-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5423-5434 ◽

Cited By ~ 47

Author(s):

Kerstin Lorz ◽

Heike Hofmann ◽

Anja Berndt ◽

Nina Tavalai ◽

Regina Mueller ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Transcriptional Activation ◽

Deletion Mutant ◽

Artificial Chromosome ◽

Open Reading Frame ◽

Wild Type Virus ◽

Wild Type ◽

Reading Frame ◽

Transcriptional Activation Domain ◽

Viral Particles

ABSTRACT We previously showed that open reading frame (ORF) UL26 of human cytomegalovirus, a member of the US22 multigene family of betaherpesviruses, encodes a novel tegument protein, which is imported into cells in the course of viral infection. Moreover, we demonstrated that pUL26 contains a strong transcriptional activation domain and is capable of stimulating the major immediate-early (IE) enhancer-promoter. Since this suggested an important function of pUL26 during the initiation of the viral replicative cycle, we sought to ascertain the relevance of pUL26 by construction of a viral deletion mutant lacking the UL26 ORF using the bacterial artificial chromosome mutagenesis procedure. The resulting deletion virus was verified by PCR, enzyme restriction, and Southern blot analyses. After infection of human foreskin fibroblasts, the UL26 deletion mutant showed a small-plaque phenotype and replicated to significantly lower titers than wild-type or revertant virus. In particular, we noticed a striking decrease of infectious titers 7 days postinfection in a multistep growth experiment, whereas the release of viral DNA from infected cells was not impaired. A further investigation of this aspect revealed a significantly diminished stability of viral particles derived from the UL26 deletion mutant. Consistent with this, we observed that the tegument composition of the deletion mutant deviates from that of the wild-type virus. We therefore hypothesize that pUL26 plays a role not only in the onset of IE gene transcription but also in the assembly of the viral tegument layer in a stable and correct manner.

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Increased L-Ornithine Production in Corynebacterium glutamicum by Overexpression of a Gene Encoding a Putative Aminotransferase

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000375124 ◽

2015 ◽

Vol 25 (1) ◽

pp. 45-50 ◽

Cited By ~ 8

Author(s):

Dai-Joong Kim ◽

Gui-Hye Hwang ◽

Ji-Na Um ◽

Jae-Yong Cho

Keyword(s):

Biosynthetic Pathway ◽

Gene Promoter ◽

Open Reading Frame ◽

Aminotransferase Activity ◽

Wild Type ◽

Gene Encoding ◽

Reading Frame ◽

Dehydrogenase Gene ◽

Putative Aminotransferase ◽

Combined Overexpression

Overexpression of the NCgl0462 open reading frame, encoding a class II aminotransferase, was studied in conjunction with other enzymes in <smlcap>L</smlcap>-ornithine biosynthesis in an <smlcap>L</smlcap>-ornithine-producing strain. Expression of the wild-type NCgl0462 open reading frame, which displayed aminotransferase activity, was amplified by placing it under the control of the glyceraldehyde 3-phosphate dehydrogenase gene promoter in the pEK0 plasmid and in the genome. <smlcap>L</smlcap>-Ornithine production in Corynebacteriumglutamicum SJC8260 harboring plasmid and the genomic NCgl0462 open reading frame was increased by 8.8 and 21.6%, respectively. In addition, the combined overexpression of the NCgl0462 open reading frame within the genome along with the mutated <smlcap>L</smlcap>-ornithine biosynthesis genes (argCJBD) placed in the pEK0 plasmid in C. glutamicum SJC8260 resulted in significant improvement in <smlcap>L</smlcap>-ornithine production (12.48 g/l for combined overexpression compared with 8.42 g/l for the control). These results suggest that overexpression of the aminotransferase-encoding NCgl0462 open reading frame plays an unequivocal role in the <smlcap>L</smlcap>-ornithine biosynthetic pathway, with overlapping substrate specificity in C. glutamicum.

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