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.     

Mutants defective in herpes simplex virus type 2 ICP4: isolation and preliminary characterization.

Vero cells were biochemically transformed with the gene encoding ICP4 of herpes simplex virus type 2 (HSV-2). These cells were used as permissive hosts to isolate and propagate HSV-2 mutants defective in this gene. Two mutants, designated hr259 and hr79, were isolated. Neither mutant grew in nontransformed Vero cells, but both grew to near wild-type levels in HSV-2 ICP4-expressing cells. hr259 contains a deletion of about 0.6 kilobases which eliminates the mRNA start site of the ICP4 gene. hr79 contains a mutation which maps by marker rescue to the portion of the ICP4 gene encoding the carboxy-terminal half of the protein. Although hr259 failed to generate any detectable ICP4 mRNA in nontransformed Vero cells, hr79 encoded an ICP4 mRNA which is wild type with respect to size. In nontransformed Vero cells infected with hr259, only ICP0, ICP6, ICP22, and ICP27 were readily detectable. In the case of hr79, a truncated form of ICP4 appeared to be made in addition to ICP0, ICP6, ICP22, and ICP27. Both hr259 and hr79 grew efficiently on cell lines transformed with the ICP4 gene of HSV-1 as evidenced by plating efficiencies and single-burst experiments. Similarly, cells transformed with the ICP4 gene of HSV-2 served as efficient hosts for the growth of d120, HSV-1 ICP4 deletion mutant.     

A mutation in the latency-related gene of bovine herpesvirus 1 inhibits protein expression from open reading frame 2 and an adjacent reading frame during productive infection

The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5′ terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.     

The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase

The DNA sequence of the gene for the fermentative yeast alcohol dehydrogenase has been determined. The structural gene contains no introns. The amino acid sequence of the protein as determined from the nucleotide sequence disagrees with the published alcohol dehydrogenase isozyme I (ADH-I) sequence for 5 of the 347 amino acid residues. At least one, and perhaps as many as four, of these differences is probably due to ADH-I protein heterogeneity in different yeast strains and not to sequencing errors. S1 nuclease was used to map the 5' and 3' ends of the ADH-I mRNA. There are two discrete, mature 5' ends of the mRNA, mapping 27 and 37 nucleotides upstream of the translation initiating ATG. These two equally prevalent termini are 101 and 91 nucleotides, respectively, downstream from a TATAAA sequence. Analysis of the 3' end of ADH-I mRNA disclosed two minor ends upstream of the major poly(A) addition site. These three ends map 24, 67, and 83 nucleotides, respectively, downstream from the translation-terminating TAA triplet. The sequence AA-TAAG is found 28 to 34 nucleotides upstream of each ADH-I mRNA poly(A) addition site. Sequence comparisons of these three 3' ends with those for four other yeast mRNAs yielded a 13-nucleotide consensus sequence to which TAAATAAGA is central. All of the known yeast poly(A) addition sites map at or near the A residue of a CTA site 25 to 40 nucleotides downstream from this consensus octamer.