Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.     

Host range determinants located on the interior of the poliovirus capsid.

The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.     

A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans.

A mutation in the genome of poliovirus type 3 that is known to reduce neurovirulence in humans similarly reduces neurovirulence in mice when incorporated into a mouse-adapted-human poliovirus recombinant. Viral recombinants with a uracil at nucleotide position 472 in the 5'-noncoding regions of their genomes are unable to replicate in the mouse brain. Viral recombinants with a cytosine at this position are neurovirulent in mice. Neurovirulence of poliovirus in mice may therefore prove to be a useful indicator of the genetic stability of new attenuating mutations created by site-directed mutagenesis.     

Sublethal doses of β-amyloid peptide abrogate DNA-dependent protein kinase activity

Accumulation of DNA damage and deficiency in DNA repair potentially contribute to the progressive neuronal loss in neurodegenerative disorders, including Alzheimer disease (AD). In multicellular eukaryotes, double strand breaks (DSBs), the most lethal form of DNA damage, are mainly repaired by the nonhomologous end joining pathway, which relies on DNA-PK complex activity. Both the presence of DSBs and a decreased end joining activity have been reported in AD brains, but the molecular player causing DNA repair dysfunction is still undetermined. β-Amyloid (Aβ), a potential proximate effector of neurotoxicity in AD, might exert cytotoxic effects by reactive oxygen species generation and oxidative stress induction, which may then cause DNA damage. Here, we show that in PC12 cells sublethal concentrations of aggregated Aβ(25-35) inhibit DNA-PK kinase activity, compromising DSB repair and sensitizing cells to nonlethal oxidative injury. The inhibition of DNA-PK activity is associated with down-regulation of the catalytic subunit DNA-PK (DNA-PKcs) protein levels, caused by oxidative stress and reversed by antioxidant treatment. Moreover, we show that sublethal doses of Aβ(1-42) oligomers enter the nucleus of PC12 cells, accumulate as insoluble oligomeric species, and reduce DNA-PK kinase activity, although in the absence of oxidative stress. Overall, these findings suggest that Aβ mediates inhibition of the DNA-PK-dependent nonhomologous end joining pathway contributing to the accumulation of DSBs that, if not efficiently repaired, may lead to the neuronal loss observed in AD.     

The murine homolog (Mph) of human herpesvirus entry protein B (HveB) mediates entry of pseudorabies virus but not herpes simplex virus types 1 and 2

A mouse member of the immunoglobulin superfamily, originally designated the murine poliovirus receptor homolog (Mph), was found to be a receptor for the porcine alphaherpesvirus pseudorabies virus (PRV). This mouse protein, designated here murine herpesvirus entry protein B (mHveB), is most similar to one of three related human alphaherpesvirus receptors, the one designated HveB and also known as poliovirus receptor-related protein 2. Hamster cells resistant to PRV entry became susceptible upon expression of a cDNA encoding mHveB. Anti-mHveB antibody and a soluble protein composed of the mHveB ectodomain inhibited mHveB-dependent PRV entry. Expression of mHveB mRNA was detected in a variety of mouse cell lines, but anti-mHveB antibody inhibited PRV infection in only a subset of these cell lines, indicating that mHveB is the principal mediator of PRV entry into some mouse cell types but not others. Coexpression of mHveB with PRV gD, but not herpes simplex virus type 1 (HSV-1) gD, inhibited entry activity, suggesting that PRV gD may interact directly with mHveB as a ligand that can cause interference. By analogy with HSV-1, envelope-associated PRV gD probably also interacts directly with mHveB during viral entry.     

Changes in rhinovirus protein 2C allow efficient replication in mouse cells

Rhinovirus type 16 was found to replicate in mouse L cells that express the viral receptor, human intercellular adhesion molecule 1 (ICAM-1). However, infection of these cells at a low multiplicity of infection leads to no discernible cytopathic effect, and low virus titers are produced. A variant virus, 16/L, was isolated after alternate passage of rhinovirus 16 between HeLa and ICAM-1 L cells. Infection of mouse cells with 16/L leads to higher virus titers, increased production of RNA, and total cytopathic effect. Three amino acid changes were identified in the P2 region of virus 16/L, and the adaptation phenotype mapped to two changes in protein 2C. The characterization of a rhinovirus host range mutant will facilitate the investigation of cellular proteins required for efficient viral growth and the development of a murine model for rhinovirus infection.     

Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of mice lacking nectin-2, a component of cell-cell adherens junctions

Nectin-2 is a cell adhesion molecule encoded by a member of the poliovirus receptor gene family. This family consists of human, monkey, rat, and murine genes that are members of the immunoglobulin gene superfamily. Nectin-2 is a component of cell-cell adherens junctions and interacts with l-afadin, an F-actin-binding protein. Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. Homozygous null males are sterile, while homozygous null females, as well as heterozygous males and females, are fertile. The production by nectin-2(-/-) mice of normal numbers of spermatozoa containing wild-type levels of DNA suggests that Nectin-2 functions at a late stage of germ cell development. Consistent with such a role, Nectin-2 is expressed in the testes only during the later stages of spermatogenesis. The structural defects observed in spermatozoa of nectin-2(-/-) mice suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis.