The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil-DNA Glycosylase

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence downstream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl β-D -thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.     

Small Noncoding RNAs Encoded within the Bovine Herpesvirus 1 Latency-Related Gene Can Reduce Steady-State Levels of Infected Cell Protein 0 (bICP0)

Following acute infection in mucosal epithelium, bovine herpes virus 1 (BHV-1) establishes lifelong latency in sensory neurons within trigeminal ganglia. The latency-related RNA (LR-RNA) is abundantly expressed in sensory neurons of latently infected calves. Expression of LR proteins is necessary for the latency reactivation cycle because a mutant virus that does not express LR proteins is unable to reactivate from latency after dexamethasone treatment. LR-RNA sequences also inhibit bICP0 expression, productive infection, and cell growth. However, it is unclear how LR-RNA mediates these functions. In this study, we identified a 463-bp region within the LR gene (the XbaI-PstI [XP] fragment) that inhibited bICP0 protein and RNA expression in transiently transfected mouse neuroblastoma cells. Small noncoding RNAs (sncRNAs) encoded within the XP fragment (20 to 90 nucleotides in length) were detected in transiently transfected mouse neuroblastoma cells. Two families of sncRNAs were cloned from this region, and each family was predicted to contain a mature microRNA (miRNA). Both miRNAs were predicted to base pair with bICP0 mRNA sequences, suggesting that they reduce bICP0 levels. To test this prediction, sequences encompassing the respective sncRNAs and mature miRNAs were synthesized and cloned into a small interfering RNA expression vector. Both sncRNA families and their respective miRNAs inhibited bICP0 protein expression in mouse neuroblastoma cells and productive infection in bovine cells. In trigeminal ganglia of latently infected calves, an sncRNA that migrated between nucleotides 20 and 25 hybridized to the XP fragment. During dexamethasone-induced reactivation from latency, XP-specific sncRNA levels were reduced, suggesting that these sncRNAs support the establishment and maintenance of lifelong latency in cattle.Bovine herpes virus 1 (BHV-1) infection leads to respiratory and genital disorders, abortion, conjunctivitis, and/or multisystemic infection in small calves (19-21, 23). Consequently, BHV-1 infections are a significant economic loss to the cattle industry. As with other Alphaherpesvirinae subfamily members, the primary site for a BHV-1 latent infection is sensory ganglionic neurons (19, 20, 23). Virus reactivation from latency can occur after stress, suggesting that corticosteroids play a role in this process.During latency, viral gene expression is restricted to the latency-related (LR) gene and open reading frame E (ORF-E) (13, 23, 35, 36). The LR gene contains two open reading frames (ORF1 and ORF2) and two reading frames (RF-B and RF-C) (24). A fraction of LR-RNA is polyadenylated and alternatively spliced in trigeminal ganglia (TG), suggesting that more than one protein is expressed (4, 5, 12). A peptide antibody directed against ORF2 recognizes a protein encoded by the LR gene (12, 17, 18). LR protein expression is necessary for the latency reactivation cycle because a mutant BHV-1 strain with three stop codons at the N terminus of ORF2 does not reactivate from latency (14, 33). Furthermore, the LR mutant virus has diminished clinical symptoms and reduced shedding of infectious virus from the eye, TG, and tonsil (14, 15, 33). Finally, the LR mutant virus induces higher levels of apoptosis in TG neurons, in part because a protein encoded by the LR gene (ORF2) inhibits apoptosis (3, 14, 15, 26, 40). Three LR proteins, including ORF2, have reduced or no expression in cells infected with the LR mutant virus (18, 27).Although proteins encoded by the LR gene are necessary for the latency reactivation cycle, non-protein coding functions within LR-RNA have also been identified. For example, the intact LR gene inhibits the ability of bICP0 to stimulate productive infection in a dose-dependent manner (1, 9). Insertion of three in-frame stop codons at the amino terminus of the first ORF within the LR gene (ORF2) inhibited bICP0 repression with an efficiency similar to that of the wild-type (wt) LR gene, suggesting that expression of an LR protein is not required (9). Since the LR gene is antisense to bICP0 coding sequences, we assumed that LR-RNA hybridized to bICP0 RNA sequences and interfered with bICP0 expression. However, we were unable to obtain data suggesting that antisense repression was the major reason why the LR gene inhibited bICP0 expression. LR gene products also inhibit mammalian cell growth (8, 38), and the cell growth-inhibitory function of the LR gene maps to a 463-bp XbaI-PstI (XP) fragment (8). Sequences within the XP region have the potential to form stem-loop secondary structures, suggesting that there are small noncoding RNAs (sncRNAs) expressed from the XP region.In this study, we demonstrated that the XP fragment efficiently inhibits bICP0 protein levels and, to a lesser extent, bICP0 RNA levels. Northern blot analysis using the XP fragment as a probe detected sncRNAs migrating between 20 and 90 nucleotides (nt). Two families of sncRNAs with the same 5′ terminus but different 3′ termini were cloned from this region. Members of these two families of sncRNAs inhibited bICP0 expression with an efficiency similar to that of the XP fragment. Each family of sncRNAs has the potential to generate a mature microRNA (miRNA). Sequences encompassing the mature miRNA also inhibited bICP0 expression in transiently transfected cells. Although the miRNA sequences have the potential to base pair with bICP0 mRNA, the miRNA sequences do not overlap bICP0 RNA sequences. Finally, LR-specific sncRNAs and miRNAs inhibited productive infection approximately 2-fold, suggesting that LR-specific sncRNAs support the establishment and maintenance of lifelong latency in cattle.     

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.     

Mutational Analysis of the Open Reading Frames in the Transposable Element Is1

IS1 is one of the smallest transposable elements found in bacteria (768 bp). It contains eight overlapping open-reading-frames (ORFs) greater than 50 codons, designated insA to insG and insB'. To determine which of the ORFs actually code for proteins involved in transposition, we have introduced amber codons into each ORF by site-directed mutagenesis which make neutral changes in the overlapping ORFs. Each mutant IS1 was then tested for its ability to mediate cointegrate formation in Su+ and Su- backgrounds. The mutant elements were also tested for trans-complementation in an IS1-free Salmonella background. Our results show that the products of the insA and insB genes are the only ones essential for cointegrate formation. We suggest that other ORFs may, however, encode accessory proteins.     

Identification of Open Reading Frames in Schizosaccharomyces pombe cDNAs

A total of 214 non-overlapping cDNA clones from Schizosaccharomycespombe were selected and completely sequenced. The clones notpreviously reported were divided into the following three groups:1) homologous to Saccharomyces cerevisiae genes (139 clones);2) homologous to genes from other organisms but not to thosefrom Sac. cerevisiae (4 clones); and 3) no similar sequences(40 clones). Among the 31 sequences identical to those in thepublic databases, 4 genes have regions corresponding to introns.Protein sequences which had homologs both in budding yeast andmammals were compared with those from Sac. cerevisiae and mammals.The search revealed that the evolutionary distances among thesespecies are similar at least with genes of this category.     

Predicting Statistical Properties of Open Reading Frames in Bacterial Genomes

An analytical model based on the statistical properties of Open Reading Frames (ORFs) of eubacterial genomes such as codon composition and sequence length of all reading frames was developed. This new model predicts the average length, maximum length as well as the length distribution of the ORFs of 70 species with GC contents varying between 21% and 74%. Furthermore, the number of annotated genes is predicted with high accordance. However, the ORF length distribution in the five alternative reading frames shows interesting deviations from the predicted distribution. In particular, long ORFs appear more often than expected statistically. The unexpected depletion of stop codons in these alternative open reading frames cannot completely be explained by a biased codon usage in the +1 frame. While it is unknown if the stop codon depletion has a biological function, it could be due to a protein coding capacity of alternative ORFs exerting a selection pressure which prevents the fixation of stop codon mutations. The comparison of the analytical model with bacterial genomes, therefore, leads to a hypothesis suggesting novel gene candidates which can now be investigated in subsequent wet lab experiments.     

Reinitiation after Translation of Two Upstream Open Reading Frames (ORF) Governs Expression of the ORF35-37 Kaposi's Sarcoma-Associated Herpesvirus Polycistronic mRNA

The Kaposi''s sarcoma-associated herpesvirus (KSHV) ORF36 protein kinase is translated as a downstream gene from the ORF35-37 polycistronic mRNA via a unique mechanism involving short upstream open reading frames (uORFs) located in the 5′ untranslated region. Here, we confirm that ORF35-37 is functionally dicistronic during infection and demonstrate that mutation of the dominant uORF restricts KSHV replication. Leaky scanning past the uORFs facilitates ORF35 expression, while a reinitiation mechanism after translation of the uORFs enables ORF36 expression.