In vivo RNA localization of I factor, a non-LTR retrotransposon, requires a cis-acting signal in ORF2 and ORF1 protein

According to the current model of non-LTR retrotransposon (NLR) mobilization, co-expression of the RNA transposition intermediate, and the proteins it encodes (ORF1p and ORF2p), is a requisite for the formation of cytoplasmic ribonucleoprotein complexes which contain necessary elements to complete a retrotransposition cycle later in the nucleus. To understand these early processes of NLR mobilization, here we analyzed in vivo the protein and RNA expression patterns of the I factor, a model NLR in Drosophila. We show that ORF1p and I factor RNA, specifically produced during transposition, are co-expressed and tightly co-localize with a specific pattern (Loc+) exclusively in the cytoplasm of germ cells permissive for retrotransposition. Using an ORF2 mutated I factor, we show that ORF2p plays no role in the Loc+ patterning. With deletion derivatives of an I factor we define an RNA localization signal required to display the Loc+ pattern. Finally, by complementation experiments we show that ORF1p is necessary for the efficient localization of I factor RNA. Our data suggest that ORF1p is involved in proper folding and stabilization of I factor RNA for efficient targeting, through Loc+ patterning, to the nuclear neighborhood where downstream steps of the retrotransposition process occur.     

Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.     

The pcd Gene Encoding Piperideine-6-Carboxylate Dehydrogenase Involved in Biosynthesis of α-Aminoadipic Acid Is Located in the Cephamycin Cluster of Streptomyces clavuligerus

Three open reading frames (ORFs) have been located downstream of cefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74,094).     

Feline calicivirus VP2 is essential for the production of infectious virions

The third open reading frame (ORF3) located at the 3′ end of the genomic RNA of feline calicivirus (FCV) encodes a small (12.2-kDa) minor structural protein of 106 amino acids designated VP2. Point mutations and deletions were introduced into an infectious FCV cDNA clone in order to evaluate the functional importance of ORF3 and its encoded protein, VP2. Deletion of the entire ORF3 sequence was lethal for the virus, and evidence was found for strong selective pressure to produce the VP2 protein. Extended deletions in the 5′ end and small deletions in the 3′ end of ORF3, as well as the introduction of stop codons into the ORF3 sequence, were tolerated by the viral replication machinery, but infectious virus could not be recovered. Infectious virus particles could be rescued from a full-length FCV cDNA clone encoding a nonfunctional VP2 when VP2 was provided in trans from a eukaryotic expression plasmid. Our data indicate that VP2, a protein apparently unique to the caliciviruses, is essential for productive replication that results in the synthesis and maturation of infectious virions and that the ORF3 nucleotide sequence itself overlaps a cis-acting RNA signal at the genomic 3′ end.     

Doc and copia instability in an isogenic Drosophila melanogaster stock

A high degree of heterogeneity and an overall increase in number of insertion sites of the mobile elements Doc and copia were revealed in one substock of an isogenic Drosophila melanogaster stock, while in two other substocks the distribution of copia sites was highly homogenous, but that of Doc sites was again heterogenous. We therefore concluded that copia was unstable in one of the substocks and Doc was unstable in all. Doc instability presumably arose earlier than copia instability. Doc and copia transpositions were directly observed in experiments with one substock. An abundance of copia insertions was revealed in the X chromosome where insertions with deleterious effects are exposed to selection in hemizygous condition. The locations of many other mobile elements (mdg1, mdg2, mdg3, mdg4, 297, B104, H.M.S. Beagle, I, P, BS, FB) were found to be conserved in each substock and did not differ between them, indicating that these mobile elements were stable. This homogeneity is a strong argument against any possibility of inadvertent contamination.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^? RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^? RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

The Structure of the Cyprinid herpesvirus 3 ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L,a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.     

The Torradovirus‐specific RNA2‐ORF1 protein is necessary for plant systemic infection

Tomato apex necrosis virus (ToANV, species Tomato marchitez virus, genus Torradovirus, family Secoviridae) causes a severe tomato disease in Mexico. One distinctive feature of torradoviruses compared with other members of the family Secoviridae is the presence of an additional open reading frame (ORF) in genomic RNA2 (denominated RNA2‐ORF1), located upstream of ORF2. RNA2‐ORF2 encodes a polyprotein that is processed into a putative movement protein and three capsid proteins (CPs). The RNA2‐ORF1 protein has homologues only amongst other torradoviruses and, so far, no function has been associated with it. We used recombinant and mutant ToANV clones to investigate the role of the RNA2‐ORF1 protein in various aspects of the virus infection cycle. The lack of a functional RNA2‐ORF1 resulted in an inability to systemically infect Nicotiana benthamiana and tomato plants, but both positive‐ and negative‐strand RNA1 and RNA2 accumulated locally in agroinfiltrated areas in N. benthamiana plants, indicating that the RNA2‐ORF1 mutants were replication competent. Furthermore, a mutant with a deletion in RNA2‐ORF1 was competent for virion formation and cell‐to‐cell movement in the cells immediately surrounding the initial infection site. However, immunological detection of the ToANV CPs in the agroinfiltrated areas showed that this mutant was not detected in the sieve elements even if the surrounding parenchymatic cells were ToANV positive, suggesting a role for the RNA2‐ORF1 protein in processes occurring prior to phloem uploading, including efficient spread in inoculated leaves.     

The ORF2 protein of hepatitis E virus binds the 5' region of viral RNA

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3' structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5' and 3' ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5' end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5' end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.     

The Crithidia fasciculata RNH1 gene encodes both nuclear and mitochondrial isoforms of RNase H

The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.     

Norwalk virus nonstructural protein p48 forms a complex with the SNARE regulator VAP-A and prevents cell surface expression of vesicular stomatitis virus G protein

Norwalk virus (NV), a reference strain of human calicivirus in the Norovirus genus of the family Caliciviridae, contains a positive-strand RNA genome with three open reading frames. ORF1 encodes a 1,789-amino-acid polyprotein that is processed into nonstructural proteins that include an NTPase, VPg, protease, and RNA-dependent RNA polymerase. The N-terminal protein p48 of ORF1 shows no significant sequence similarity to viral or cellular proteins, and its function in the human calicivirus replication cycle is not known. The lack of sequence similarity to any protein in the public databases suggested that p48 may have a unique function in the NV replication cycle or, alternatively, may perform a characterized function in replication by a unique mechanism. In this report, it is shown that p48 displays a vesicular localization pattern in transfected cells when fused to the fluorescent reporter EYFP. A predicted transmembrane domain at the C terminus of p48 was not necessary for the observed localization pattern, but this domain was sufficient to redirect localization of EYFP to a fluorescent pattern consistent with the Golgi apparatus. A yeast two-hybrid screen identified the SNARE regulator vesicle-associated membrane protein-associated protein A (VAP-A) as a binding partner of p48. Biochemical assays confirmed that p48 and VAP-A interact and form a stable complex in mammalian cells. Furthermore, expression of the vesicular stomatitis virus G glcyoprotein on the cell surface was inhibited when cells coexpressed p48, suggesting that p48 disrupts intracellular protein trafficking.     

The transmembrane domain of the severe acute respiratory syndrome coronavirus ORF7b protein is necessary and sufficient for its retention in the Golgi complex

The severe acute respiratory syndrome coronavirus (SARS-CoV) ORF7b (also called 7b) protein is an integral membrane protein that is translated from a bicistronic open reading frame encoded within subgenomic RNA 7. When expressed independently or during virus infection, ORF7b accumulates in the Golgi compartment, colocalizing with both cis- and trans-Golgi markers. To identify the domains of this protein that are responsible for Golgi localization, we have generated a set of mutant proteins and analyzed their subcellular localizations by indirect immunofluorescence confocal microscopy. The N- and C-terminal sequences are dispensable, but the ORF7b transmembrane domain (TMD) is essential for Golgi compartment localization. When the TMD of human CD4 was replaced with the ORF7b TMD, the resulting chimeric protein localized to the Golgi complex. Scanning alanine mutagenesis identified two regions in the carboxy-terminal portion of the TMD that eliminated the Golgi complex localization of the chimeric CD4 proteins or ORF7b protein. Collectively, these data demonstrate that the Golgi complex retention signal of the ORF7b protein resides solely within the TMD.     

Host RAB11FIP5 protein inhibits the release of Kaposi’s sarcoma-associated herpesvirus particles by promoting lysosomal degradation of ORF45

Open reading frame (ORF) 45 is an outer tegument protein of Kaposi’s sarcoma-associated herpesvirus (KSHV). Genetic analysis of an ORF45-null mutant revealed that ORF45 plays a key role in the events leading to the release of KSHV particles. ORF45 associates with lipid rafts (LRs), which is responsible for the colocalization of viral particles with the trans-Golgi network and facilitates their release. In this study, we identified a host protein, RAB11 family interacting protein 5 (RAB11FIP5), that interacts with ORF45 in vitro and in vivo. RAB11FIP5 encodes a RAB11 effector protein that regulates endosomal trafficking. Overexpression of RAB11FIP5 in KSHV-infected cells decreased the expression level of ORF45 and inhibited the release of KSHV particles, as reflected by the significant reduction in the number of extracellular virions. In contrast, silencing endogenous RAB11FIP5 increased ORF45 expression and promoted the release of KSHV particles. We further showed that RAB11FIP5 mediates lysosomal degradation of ORF45, which impairs its ability to target LRs in the Golgi apparatus and inhibits ORF45-mediated colocalization of viral particles with the trans-Golgi network. Collectively, our results suggest that RAB11FIP5 enhances lysosome-dependent degradation of ORF45, which inhibits the release of KSHV particles, and have potential implications for virology and antiviral design.