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There has been a heightened awareness of an increased risk of suicidality among individuals with autism spectrum disorder (ASD) due to high rates of suicidal ideation (SI) in this population (11-66%). The current study investigated the rate of parent-endorsed SI and associated clinical features in 48 youths with ASD (Age; M: 12.97 years, SD: 2.33). SI was endorsed in 18.75% of participants. Youth with SI exhibited significantly higher levels of affective problems, externalizing problems, feelings of humiliation and rejection, and symptoms related to perfectionism. Results indicate that co-occurring mental health problems are associated with suicidal ideation and provide relevant targets for psychotherapeutic intervention. This preliminary study in a modest sample suggests the value of further research in larger samples to replicate and generalize these findings.

There are many empirically supported treatment options for anxiety disorders in the literature; however, there has been a lack of options for the treatment of anxiety disorders among individuals with intellectual disabilities. This chapter reviews evidence-based treatment tools for anxiety for individuals with various levels of intellectual disability. Behavioral treatment components geared toward all severity levels are reviewed, including exposure and reinforcement techniques. Additionally, modified versions of cognitive behavioral treatment components are discussed for milder severity levels, which have been a growing part of the literature.

Transcripts of most intron-bearing cellular genes must be processed by the splicing machinery in order to efficiently accumulate and gain access to the cytoplasm. However, we found that herpes simplex virus induces cytoplasmic accumulation of both spliced and unspliced polyadenylated α-globin RNAs in infected HeLa cells. Accumulation of the unspliced RNA required the immediate-early protein ICP27, and ICP27 was sufficient (in combination with ICP4) to produce this effect in a transient-transfection assay. However, expression of ICP27 did not markedly alter the levels of fully spliced α-globin transcripts in infected cells. These data demonstrate that the previously documented effects of ICP27 on the cellular splicing apparatus do not greatly inhibit splicing of α-globin RNA and argue that ICP27 induces a splicing-independent pathway for α-globin RNA accumulation and nuclear export.

The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosylase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia virus ts4149-infected cells in order to assess the recombination events that allowed virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.

Myxoma virus is an infectious poxvirus pathogen that induces a virulent systemic disease called myxomatosis in European rabbits. The disease is rapidly and uniformly fatal to susceptible rabbits and is characterized by generalized dysfunction of cellular immunity and multiple interruptions of the host cytokine network. A number of virus genes are classified as virulence factors because virus constructs bearing targeted gene disruptions induce attenuated disease symptoms. Many of these genes encode proteins that interact directly with effector elements of the host immune system. Included among these immunosubversive viral proteins are secreted mimics of host ligands or regulators (virokines) and homologues of cellular cytokine receptors (viroceptors). Five examples of these immune modulator proteins encoded by myxoma virus are reviewed: (1) myxoma growth factor, a member of the epidermal growth factor ligand superfamily; (2) SERP-1, a secreted serine proteinase inhibitor; (3) M11L, a receptor-like surface protein; (4) T2, a tumor necrosis factor receptor homologue; and (5) T7, an interferon-γ receptor homologue. The origin of viral strategies designed to subvert immune regulation by host cytokines is considered in the context of the biology of myxoma virus within immunocompetent hosts. J. Leukoc. Biol. 57: 731–738; 1995.

Available evidence suggests that one or more late viral gene products are involved in processing poxvirus replicative intermediates into mature progeny hairpin-terminated genomes. Cloned versions of the Shope fibroma virus (SFV) replicated telomere in the inverted repeat configuration were used as substrates to assay lysates from poxvirus-infected cells for protein fractions that participate in the resolution of the circular substrate plasmid into a linear minichromosome with viral hairpin termini. An activity in a crude protein fraction obtained from vaccinia virus-infected cells at late times during the replicative cycle was capable of accurately resolving all poxviral inverted repeat replicative intermediates tested. The resolved linear products are identical to the products of in vivo resolution and possessed symmetrical nicks which mapped at the borders of the inverted repeat sequence. Strand-specific nicks were also identified, which mapped within the telomere resolution target sequence known to be required for telomere resolution in vivo. The resolving activity that we have identified is specific to virus-infected cells at late times during replication and cleaves cloned poxviral telomeric substrates in a fashion expected of a classic Holliday junction-resolving enzyme in addition to possessing a telomere resolution target-specific nicking activity. Although a Holliday junction-resolving activity would also be expected to play a role in the recombination induced by poxvirus infection, the appearance of the activity described here only after the commencement of viral late protein synthesis suggests that it functions strictly at late times. Other non-viral Holliday junction analogs can also be cleaved by this extract, suggesting that this component of the resolution activity may also play a role in other viral processes that require cleavage of a branched DNA structure. Thus, we have identified a poxviral activity that may be a part of a protein complex which resolves concatemeric replicative intermediates of viral DNA as well as participate in general recombination late during infection.

Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of gene expression that modulates the synthesis and maturation of viral and cellular mRNAs. Processes that are affected by ICP27 include transcription, pre-mRNA splicing, polyadenylation, and nuclear RNA export. We have examined how ICP27 influences the expression of the essential HSV tegument protein and transactivator of immediate-early gene expression VP16. We monitored the effects of ICP27 on the levels, nuclear export, and polyribosomal association of VP16 mRNA and on the amount and stability of VP16 protein. Deletion of ICP27 reduced the levels of VP16 mRNA without altering its nuclear export or the stability of the encoded protein. However, the translational yield of the VP16 mRNA produced in the absence of ICP27 was reduced 9- to 80-fold relative to that for wild-type infection, suggesting a defect in translation. In the absence of ICP27, the majority of cytoplasmic VP16 mRNA was not associated with actively translating polyribosomes but instead cosedimented with 40S ribosomal subunits, indicating that the translational defect is likely at the level of initiation. These effects were mRNA specific, as polyribosomal analysis of two cellular transcripts (glyceraldehyde-3-phosphate dehydrogenase and β-actin) and two early HSV transcripts (thymidine kinase and ICP8) indicated that ICP27 is not required for efficient translation of these mRNAs. Thus, we have uncovered a novel mRNA-specific translational regulatory function of ICP27.

Herpes simplex virus (HSV) is the prototypical member of the Herpesviridae, a large group of enveloped nuclear DNA viruses that infect a wide range of metazoan organisms. Like all herpesviruses, HSV displays both lytic and latent modes of interaction with its natural human host (reviewed in reference 46). The HSV lytic cycle involves a complex genetic program encompassing a variety of transcriptional and posttranscriptional controls (reviewed in references 13, 46, and 63): expression of most cellular genes is strongly suppressed, and three temporal classes of viral genes are sequentially activated in a regulatory cascade. Five immediate-early (IE) genes are expressed first, through the transactivation function of the virion protein VP16 in combination with cellular factors. Four of the IE gene products (ICP0, ICP4, ICP22, and ICP27) are nuclear regulatory proteins that orchestrate the timely expression of the early (E) and late (L) genes. The IE protein ICP27 is essential for the viability of the virus in cultured cells, and ICP27 homologs are present in all of the mammalian and avian herpesvirus genomes that have been characterized to date. ICP27 plays a fundamental and multifunctional role in the viral life cycle that has yet to be completely defined. HSV type 1 (HSV-1) ICP27 null mutants display reduced levels of some E and most L mRNAs and are defective in viral DNA replication (12, 26, 30, 41, 42, 48, 53, 59). In addition, they fail to efficiently suppress cellular gene expression (16, 18, 48). The modes of action of ICP27 in these various processes are not completely clear; however, it is becoming increasingly apparent that ICP27 functions primarily to modulate the posttranscriptional processing and transport events that are required to convert nuclear primary transcripts into functional mRNA molecules in the cytoplasm. It has been recognized for some time that ICP27 can activate or repress the expression of reporter genes driven by HSV promoters in transient cotransfection assays (17, 30, 41, 42, 45, 53, 58). Although it was initially thought that the activation and repression was at the transcriptional level, it is now clear that ICP27 exerts these effects at least in part by modulating the processes of polyadenylation and splicing (18, 27, 28, 37, 39, 51, 52). Rather than being promoter dependent, activation of gene expression results from the enhancement of the selection and cleavage of weak poly(A) sites (27–29, 52). The repression function correlates with the presence of introns in the reporter genes (52), a finding that led to the view that ICP27 represses expression of intron-bearing genes by inhibiting RNA splicing. A significant amount of data has supported the notion that ICP27 impairs or modulates splicing: (i) ICP27 colocalizes with and redistributes snRNPs in HSV-infected cell nuclei (37); (ii) ICP27 coimmunoprecipitates with splicing factors that react with anti-Sm antisera and appears to alter the phosphorylation status of some of these proteins (50); and (iii) nuclear extracts prepared from cells infected with wild-type HSV carry out in vitro splicing reactions less efficiently than those prepared from uninfected cells, and this reduction requires ICP27 (18). It has been suggested that inhibition of splicing by ICP27 is responsible for the delayed shutoff of cellular gene expression that occurs during HSV infection (52). This is an appealing idea because unlike cellular genes, the majority of HSV genes do not contain introns, and thus HSV gene expression would be relatively resistant to inhibition. Consistent with this hypothesis, ICP27 mutants fail to induce the decline in the levels of cellular mRNAs characteristic of infection with wild-type HSV (16, 18). ICP27 is a nuclear/cytoplasmic shuttling protein (32, 38, 49, 55) and has been shown to bind RNA through a region rich in arginine and glycine residues, the RGG box (33). A model has emerged recently suggesting that these activities mediate the cytoplasmic accumulation of intronless viral L RNAs in a fashion similar to that for the human immunodeficiency virus (HIV) Rev protein (49, 55, 57). ICP27 may also play a role in increasing the stability of certain RNAs (1). In keeping with its multifunctional role in viral gene expression, the 512-residue ICP27 protein is composed of multiple functional regions that confer several possibly independent properties on the protein. For example, ICP27's nuclear/cytoplasmic shuttling ability is conferred by a leucine-rich nuclear export signal (NES) at the N terminus similar to that of the HIV Rev protein (49), in combination with multiple nuclear localization signals (NLS), including a strong NLS localized to amino acids 110 to 137 (31). The RGG box corresponds to residues 138 to 152 (33). The C-terminal half of the molecule has been implicated in the above-mentioned effects on polyadenylation (the activation function) and splicing (the repression function) (17, 30, 42, 45), although residues in the N terminus also appear to contribute to activation (45) and repression (44). Not surprisingly in view of the complex functions it fulfills, ICP27 has been shown to interact with numerous other viral and cellular proteins. Physical interactions with the main viral transactivator, ICP4, have been documented (36). Furthermore, it has recently been shown that ICP27 can self-associate (65). Interactions with proteins of the splicing apparatus (50), hnRNP K and casein kinase 2 (61), have also been demonstrated, all of which may contribute in various ways to the functions of ICP27. We have been investigating how ICP27 affects mRNA processing and nuclear export of the transcript encoded by the cellular α-globin gene. Normally silent in cells of nonerythroid lineage, the α-globin gene is induced during HSV infection by the actions of the IE proteins ICP0, ICP4, and ICP22, leading to accumulation of correctly initiated RNAs (6). We have recently reported that ICP27, while having little effect on the levels of spliced α-globin RNA, causes cytoplasmic accumulation of unspliced α-globin pre-mRNA (5). This observation was surprising, for two reasons. First, a large body of evidence demonstrates that transcripts of most intron-bearing cellular genes must be processed by the splicing apparatus in order to access the nuclear export machinery (2, 7–9, 14, 19, 20, 22, 25, 35, 47). The process of splicing itself appears to be required, as evidenced by the finding that cDNA copies of many intron-bearing genes fail to direct the accumulation of stable cytoplasmic RNA, and this defect can be rescued by placing a heterologous intron in the transcription unit (14). Thus, our finding that ICP27 promotes cytoplasmic accumulation of unspliced α-globin RNA suggested that ICP27 provides a novel splicing-independent pathway for RNA export. Second, previous studies by other investigators have been interpreted to indicate that ICP27 induces nuclear retention of intron-bearing transcripts of the HSV genes encoding ICP0 and UL15 (18, 39, 49). This effect, ascribed to a global inhibition of splicing mediated by ICP27, was taken to suggest that the RNA transport function of ICP27 distinguishes between transcripts arising from intron-bearing and intronless genes and is capable of transporting only the latter. In contrast, our data were more compatible with the hypothesis that ICP27 stimulates splicing-independent transport of a specific subset of RNAs, irrespective of the presence or absence of introns. Our hypothesis that ICP27 causes the accumulation of α-globin pre-mRNA by inducing a splicing-independent RNA transport system (rather than by inhibiting the process of splicing per se) raised the possibility that accumulation of unspliced α-globin RNA could be uncoupled by mutation from the previously described transrepression function of ICP27, which has been attributed to direct inhibition of splicing. In addition, the contrast between our results and those previously reported for intron-bearing transcripts of the HSV ICP0 and UL15 genes suggested that ICP27 can discriminate between different intron-bearing RNAs. Here we present the results of experiments that test these predictions, by examining the effects of a large panel of ICP27 mutations on the processing and transport of α-globin and ICP0 RNAs.

A plasmid was constructed in which a single guanine residue was replaced with either O6-methylguanine or O6-ethylguanine, two of the DNA adducts formed by carcinogenic alkylating agents. The vectors were introduced in parallel into a pair of Chinese hamster ovary cells, in which one member of the pair was deficient in the repair enzyme O6-alkylguanine-DNA alkyltransferase (mex-) and the other was proficient in this activity (mex+). The vectors integrated into and replicated within the respective host genomes. After intrachromosomal replication, the DNA sequence in the vicinity of the originally adducted site of each integrated vector was amplified from the host genome by using the polymerase chain reaction and was analyzed for mutations. High levels of mutation were observed from the O6-methylguanine- and O6-ethylguanine-containing vectors replicated in mex- cells (approximately 19% for O6-methylguanine and approximately 11% for O6-ethylguanine). DNA sequencing revealed the induced mutations to be almost exclusively G----A transitions. By contrast, little or no mutagenesis was detected when the adducted vectors were introduced into mex+ cells, indicating the significant role of the O6-alkylguanine-DNA alkyltransferase in the repair of O6-methylguanine and O6-ethylguanine in these mammalian cells.

A shuttle vector, pKE15, was constructed for investigating the mechanisms by which single carcinogen-DNA adducts induce mutations in mammalian cells. pKE15 contains the SV40 origin of replication, the neomycin resistance gene, SV40 polyadenylation sequences and the pML2 origin of replication. Transfection of pKE15 into CHO cells established the G418-resistant phenotype; the frequency of G418-resistant clones was approximately 10(-4), a value that is similar to those obtained with other SV40-based vectors expressing the neomycin resistance gene. A tetranucleotide containing O6-methylguanine, a DNA adduct formed by carcinogenic alkylating agents, was incorporated into a 4-base gap positioned in the center of a PstI site. The tetranucleotide containing the adduct was physically mapped to a 14-base-pair region of the shuttle vector that included the ligation target, the PstI site. It was incorporated approximately equally into either of the complementary strands of the shuttle vector. The ligation efficiency of the tetranucleotide into the gapped genome was approximately 100% and was independent of the concentration of tetranucleotide used at concentrations ranging over one order of magnitude. The potential applications of the site-specifically modified genome for establishing the mutagenic fate of O6-methylguanine in repair-proficient and -deficient CHO cells are discussed.