Late L2 Research Articles

The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels.

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3' untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.

Aedes albopictus (Diptera: Culicidae): an experimental and natural host of Dirofilaria immitis (Filarioidea: Onchocercidae) in Florida, U.S.A.

Females of an Aedes albopictus (Skuse) colony from southeastern Florida, U.S.A., ingested low (22.9 +/- 3.2 mg/Female) and high (243.2 +/- 37.6 mf/Female) numbers of microfilariae from a dog infected with Dirofilaria immitis (Leidy). High mortality of females occurred during the first 4 d after infection regardless of the number of microfilariae ingested; daily mortality was almost negligible during 5-15 d after infection. Percentage of survival 15 d after infection was higher (63%) in females that ingested low numbers of microfilariae than those (15%) that ingested high numbers of microfilariae. The development of most of the D. immitis larvae was arrested in late L1 stage with some of the L1 stage larvae becoming melanized intracellularly in the Malpighian tubule cells of Ae. albopictus. Fifteen days after infection, development of D. immitis to the infective L3 stage occurred in only 10.9% of the surviving F1 and F2 Ae. albopictus that ingested low numbers of microfilariae, but in 94% of Anopheles quadrimaculatus Say ingesting similar numbers of microfilariae, as a control. Females of Ae. albopictus ingesting high numbers of microfilariae had more surviving females with L3 than those ingesting low numbers of microfilariae. The number of L3 larvae in the Malpighian tubules, hemocoel, head capsule and proboscis ranged from 1 to 37 per female, indicating the potential of Ae. albopictus to transmit D. immitis. Development of D. immitis larvae was not affected by co-infection with Ascogregarina taiwanensis (Lien & Levine), although both parasites infect the Malpighian tubules, the first intracellularly and the second extracellularly. After one generation of selection, a strain of Ae. albopictus susceptible to D. immitis developed 2.5 times more L3 than the parent strain. These results show that a small portion of the natural population of Ae. albopictus is susceptible to infection with D. immitis and that susceptibility may be increased rapidly by selection. The presence of developing Dirofilaria sp. larvae in the Malpighian tubules of field-caught females indicated that Ae. albopictus may be infected naturally with D. immitis in Florida.

Input and SLA: Adults' Sensitivity to Different Sorts of Cues to French Gender

This article investigates experimentally if beginner adult learners, given auditory stimuli, are equally likely to represent French gender subclasses in terms of phonological, morphosyntactic, and/or semantic representations. Eighty‐eight adult English speakers learned patterned [Det + N]French+ translation equivalentEnglish lists. Analyses of results reveal that participants more readily cognized “natural” semantic and morphological patterns; these same lists lent themselves to generalization. The results demonstrate that for this group of learners, the construct of input for gender learning emerges through the construction of abstract knowledge representations, apparently on the basis of prior knowledge, and not solely from objective patterns in the speech signal. They provide support for theories of linguistic cognition involving mediating structural representations, as well as learning theories in which conceptual information can guide grammatical development.

Completion of the DNA sequence of mouse adenovirus type 1: Sequence of E2B, L1, and L2 (18–51 map units)

The DNA sequence of 9991 nt, corresponding to 18–51 map units of mouse adenovirus type 1 (MAV-1), was determined, completing the sequence of the Larsen strain of MAV-1. The length of the complete MAV-1 genome is 30 946 nucleotides, consistent with previous experimental estimates. The 18–51 map unit region encodes early region 2B proteins necessary for adenoviral replication as well as late region L1 and L2 structural and packaging proteins. Sequence comparison in this region with human adenoviruses indicates broad similarities, including colinear preservation of all recognized open reading frames (ORFs), with highest amino acid identity occurring in the DNA polymerase and polypeptide III (penton base subunit) ORFs. Virus-assciated (VA) RNA is not encoded in the region where VA RNAs are found in the human adenoviruses, between E2B and L1, nor is it encoded anywhere in the entire MAV-1 genome. The MAV-1 polypeptide III lacks the arginin-glycine-aspartic acid (RGD) motif which is involved in an association with cell-surface integrins. Only one RGD sequence is found in an identified coding region in the entire MAV-1 genome. Similar to the porcine adenovirus, this RGD sequence is found in the C-terminus of the MAV-1 fiber protein.

Promoter activity of sequences located upstream of the human papillomavirus types of 16 and 18 late regions.

The regulation of human papillomavirus (HPV) late gene expression is difficult to analyse because the late proteins L1 and L2 are only produced in the upper layers of terminally differentiated keratinocytes. However, for the minor capsid protein L2 of HPV types 1, 6, 11 and 16, rare mRNAs or cDNAs starting 3' of the E5 open reading frame (ORF) were previously described. In order to analyse whether the DNA region preceding the late ORFs (late upstream region, LUR) of HPV-16 and HPV-18 has promoter activity, transient transfection assays employing luciferase reporter constructs were performed. The results show that the LUR of HPV-16 and HPV-18 exhibits an orientation-dependent promoter activity in different cells. By analysing 3'-deletion mutants of the HPV-16 LUR, we identified 78 bp within the sequence between the E5 and L2 ORFs to be critical for the promoter activity. Furthermore, the analysis of a 5'-deletion mutant revealed a negative cis-regulatory element located within the E2 ORF. The HPV-16 early poly(A) signal is located downstream of the critical promoter region. Inactivation of this element by site-directed mutagenesis strongly enhanced luciferase activity. However, mutation of two potential TATA-binding protein (TBP) sites located within the critical promoter region did not abolish the activity. Altogether, these data indicate the possibility of a TATA-less promoter in the HPV-16 and HPV-18 LURs. Together with the early poly(A) signal, this potential promoter might be involved in the differentiation-dependent regulation of late gene expression.

Efficient expression of the human papillomavirus type 16 L1 protein in epithelial cells by using Rev and the Rev-responsive element of human immunodeficiency virus or the cis-acting transactivation element of simian retrovirus type 1.

Production of the human papillomavirus (HPV) late gene products L1 and L2 is limited to terminally differentiated keratinocytes. Here, we demonstrate that mRNA encoding the HPV-16 L1 capsid protein contains cis-acting RNA elements that inhibit expression at the posttranscriptional level. While cytoplasmic L1 mRNA is detectable in transfected HeLa cells, L1 protein is not produced. We have identified at least one major inhibitory element that is located within the L1 open reading frame, whereas another negative element had been reported to lie in the 3'-untranslated region of L1. The presence of these elements may explain the lack of HPV late gene expression in undifferentiated epithelial cells. Efficient production of HPV-16 L1 could be achieved with posttranscriptional regulatory elements of human immunodeficiency virus type 1 or simian retrovirus type 1. L1 protein was expressed in the presence of human immunodeficiency virus type 1 Rev from hybrid mRNAs containing the RNA binding site for Rev (Rev-responsive element). In addition, we have achieved efficient expression of L1 from hybrid mRNAs containing a cis-acting transactivation element from simian retrovirus type 1. Our data show that HPV-16 L1 protein production is regulated posttranscriptionally. This regulated expression may allow virus production in terminally differentiated epithelial cells and is probably a conserved and important mechanism for HPV expression.

Relation between skin cancer, humoral responses to human papillomaviruses, and HLA class II molecules in renal transplant recipients.

Human papillomaviruses (HPV), especially the epidermodysplasia verruciformis (EV)-associated HPV 5, 8, 14, 17, 20, and 47, are thought to play a role in the pathogenesis of some skin cancers in recipients of renal allografts. MHC class I and class II genes are involved in the cellular immune response to viral and tumor Ag. Little is known about humoral responses to HPV in recipients with and without skin cancer. We investigated the prevalence of antibodies to the early (E) protein E7 and the major capsid late (L) protein L1 of HPV 8. In addition, we studied the association of HLA class II molecules with these antibody responses. The E7 and L1 open reading frames of HPV 8 were bacterially expressed as beta-galactosidase fusion proteins, which were purified by preparative gel electrophoresis. Serum samples from 36 renal transplant recipients with and 91 recipients without skin cancer were screened for the presence of IgG and IgM antibodies to HPV 8 E7 and L1, by Western blot analysis. The detection of anti-HPV 8 L1 antibodies represents the immune response to HPV 8 and possibly other EV-associated HPV, because cross-reactivity between the representatives of this HPV subgenus can occur. The antibody responses to HLA Ag were used as controls. Recipients who had IgM antibodies but no IgG antibodies to L1 of HPV 8 (patients with no apparent class switch from IgM to IgG) had skin cancer in 50% of cases, whereas recipients who produced IgG antibodies (patients with an apparently good humoral response to L1 of HPV 8) had skin cancer in only 18% of cases. The estimated relative risk of skin cancer in recipients with no class switch, compared with the risk in those with a good humoral response, was 4.5 (95% confidence interval, 1.1 to 18.1). We found no association between the antibody response to HLA Ag and the occurrence of skin cancer. A strong linkage between the absent class switch of antibody production in response to L1 of HPV 8 and HLA-DR7 was observed (relative risk, 26.2). Renal transplant recipients who have no apparent class switch from IgM to IgG production in response to Ag encoded by L1 of HPV 8 or possibly other EV-associated HPV are at an increased risk of skin cancer. The association with HLA-DR7 indicates a genetic control of skin cancer development or regression, involving genes in the class II region of the MHC.

Phylogenetic analysis of 48 papillomavirus types and 28 subtypes and variants: a showcase for the molecular evolution of DNA viruses.

Papillomaviruses are attractive models for studying the molecular evolution of DNA viruses because of the large number of isolates that exhibit genomic diversity and host species and tissue specificity. To examine their relationship, we selected two amino acid sequences, one of 52 residues within the early gene E1 and the other of 44 residues within the late gene L1, which allowed insertion- and deletion-free alignment of all accessible papillomavirus sequences. We constructed phylogenetic trees from the amino acid and corresponding nucleotide sequences from 28 published and 20 newly determined animal and human papillomavirus (HPV) genomic sequences by using distance matrix, maximum-likelihood, and parsimony methods. The trees agreed in all important topological aspects. One major branch with two clearly separated clusters contained 11 HPV types associated with epidermodysplasia verruciformis. A second major branch had all the papillomaviruses involved in genital neoplasia and, in distant relationship, the cutaneous papillomaviruses HPV type 2a (HPV-2a), HPV-3, and HPV-10 as well as the "butcher's" papillomavirus HPV-7 and two simian papillomaviruses. Four artiodactyl (even-toed hoofed mammal) papillomaviruses, the cottontail rabbit papillomavirus, and avian (chaffinch) papillomavirus type 1 formed a third major branch. Last, four papillomaviruses exhibited little affinity to any of these three branches; these were the cutaneous types HPV-1a, HPV-4, and HPV-41 and B-group bovine papillomavirus type 4. The phylogeny suggests that some branches of papillomavirus evolution are restricted to particular target tissues and that a general process of long-term papillomavirus-host coevolution has occurred. This latter hypothesis is still conjectural because of bias in the current data base for human types and the paucity of animal papillomavirus sequences. The comparison of evolutionary distances for the most closely related types with those of 28 subtypes and variants of HPV-2, HPV-5, HPV-6, HPV-16, and HPV-18 supports the type as a natural taxonomic unit, with subtypes and variants being expressions of minor intratype genomic diversity similar to that found in the natural populations of all biological species. An exception to this seems to be HPV-2c, which has an evolutionary distance from HPV-2a of the intertype magnitude and may eventually have to be regarded as a distinct type. We describe an experimental approach that estimates the taxonomic and phylogenetic positions of newly identified papillomaviruses without viral isolation and complete genomic sequencing.(ABSTRACT TRUNCATED AT 400 WORDS)

Surface-associated antigens of Brugia malayi L2 and L3 parasites during vector-stage development

Surface and metabolic labeling procedures were used to characterize the composition and the time of expression of Brugia malayi L2 and L3 surface-associated molecules as the larvae develop within the mosquito vector. Larvae were harvested from mosquito tissues at 5 (early L2), 8(late L2) and 11 (l3) days post-infection and labeled with 125I-Iodo-Gen. The results of one-dimensional analysis showed that there is a progressive increase in the complexity of peptides associated with the surface of developing larvae, culminating in the expression of 7 major labeled components on L3s. Both L2 and L3 parasites have surface-associated components of 42,35,33,19 and 17 kDa. Between days 8 and 11 of development in the insect vector, Brugia malayi undergoes the L2 to L3 molt and acquires additional major immunogenic peptides of 40 and 22 kDa. Two-dimensional analyses of extracts from 125I-labeled L2s and L3s revealed that the major 35-,33-,19- and 17-kDa molecules are part of a peptide complex that forms a ‘ladder’ between 17 and 150kDa. To gain information on the times during which the major surface-associated molecules are produced by the parasite, larvae were labeled with [ 35]methionine either in situ as they developed within the mosquito or during culture after exiting the vector For in situ labeling. [ 35S]methionine was introduced into the hemolymph of infected mosquitoes by micro-injection at days 2, 5 and 8 post-infection and larvae were allowed to develop for an additional 3 days. The results of 1- and 2-dimensional analyses of [ 35S]methionine-labeled extracts from vector-stage or post-vector-stage larvae indicate that the molecules associated with the surface of B. malayi L3s are synthesized between day 5 and day 11 of development in the insect host. Immediately after the larvae exit the vector, the synthesis of the 40 and 22-kDa peptides is drastically reduced or terminated.

Talker and listener effects on degree of perceived foreign accent

Four experiments were carried out to examine listener- and talker-related factors that may influence degree of perceived foreign accent. In each, native English listeners rated English sentences for degree of accent. It was found that degree of accent is influenced by range effects. The larger the proportion of native (or near-native) speakers included in a set of sentences being evaluated, the more strongly accented listeners judged sentences spoken by non-native speakers to be. Foreign accent ratings were not stable. Listeners judged a set of non-native-produced sentences to be more strongly accented after, as compared to before, they became familiar with those sentences. One talker-related effect noted in the study was the finding that adults' pronunciation of an L2 may improve over time. Late L2 learners who had lived in the United States for an average of 14.3 years received significantly higher scores than late learners who had resided in the United States for 0.7 years. Another talker-related effect pertained to the age of L2 learning (AOL). Native Spanish subjects with an AOL of five to six years were not found to have an accent (i.e., to receive significantly lower scores than native English speakers), whereas native Chinese subjects with an average AOL of 7.6 years did have a measurable accent. The paper concludes with the presentation of several hypotheses concerning the relationship between AOL and degree of foreign accent.

An element in the bovine papillomavirus late 3' untranslated region reduces polyadenylated cytoplasmic RNA levels

Expression of the two bovine papillomavirus type 1 (BPV-1) late genes, L1 and L2, coding for the two capsid proteins, is limited to terminally differentiated keratinocytes in bovine fibropapillomas. This pattern of expression is determined both by the activity of the late promoter and by the inhibition of late region expression in less well differentiated cells. Inhibition of L1 and L2 mRNA production in nonpermissive cells must occur since the late region potentially could be transcribed from early region promoters. Nuclear runoff analysis of the late region has demonstrated that up to 95% of transcripts which are initiated in the early region in nonpermissive cells terminate within the late region upstream of the late polyadenylation site (C. C. Baker and J. Noe, J. Virol. 63:3529-3534, 1989). However, very few of the primary transcripts which include the late polyadenylation site are processed into mRNA. In this study, we have used expression vectors to characterize an inhibitory element active in nonpermissive cells which is located in the late 3' untranslated region (3'UTR). While the late polyadenylation site is functional in these cells, a 53-bp element in the late 3'UTR reduces levels of polyadenylated cytoplasmic RNA. This element inhibited chloramphenicol acetyltransferase (CAT) expression 6- to 10-fold when cloned in the sense orientation into the 3'UTR of a CAT expression vector. No block to expression was seen when the fragment was cloned immediately downstream of the poly(A) site, in an intron upstream of the CAT coding sequence, or in an antisense orientation in the 3'UTR. When the same fragment was deleted from a BPV-1 L1 expression vector, a sixfold increase in mRNA levels was seen. Actinomycin D chase experiments using BPV-1 L1 expression vectors indicated that the element does not destabilize cytoplasmic polyadenylated RNA. Therefore, the element must act before the mature mRNA reaches the cytoplasm. The data presented are consistent with effects on nuclear stability and/or inhibition of polyadenylation or nuclear transport.

Control of adenovirus alternative RNA splicing: effect of viral DNA replication on RNA splice site choice

The primary transcripts of most adenovirus transcription units are processed into multiple, alternatively spliced mRNAs. The relative concentrations of such differentially processed mRNAs changes during the infectious cycle. The factors that control this temporal shift in mRNA abundance have not yet been characterized. In the experiments presented here we have examined mRNA synthesis from three viral transcription units: two early regions E1a and E1b, and late region L1. We show that viral DNA replication plays a key role in the control of cytoplasmic mRNA expression from these regions. In the absence of efficient late protein synthesis, viral DNA replication was sufficient to induce a substantial fraction of the E1a, E1b and L1 transcripts to shift from the early to the late pattern of mRNA structure. The shift was not complete under the conditions used, suggesting that viral proteins, although not essential for the process, play an important regulatory role. The requirement for late viral protein synthesis differed between the three transcription units examined. This dependence was most pronounced for correct L1 mRNA production. Viral DNA replication was sufficient to trigger a significant shift in L1 alternative 3' splice site selection. However, in the absence of late translation the L1 pre-mRNA was aberrantly spliced.

Negative regulatory sequences in the lin-14 3'-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development.

The heterchronic gene lin-14 controls the temporal sequence of developmental events in the Caenorhabditis elegans postembryonic cell lineage. It encodes a nuclear protein that normally is present in most somatic cells of late embryos and L1 larvae but is absent at later stages. Two lin-14 gain-of-function mutations delete 3'-untranslated sequences causing an inappropriately high level of the lin-14 nuclear protein late in development. These mutations identify a negative regulatory element that controls the formation of the lin-14 protein temporal gradient. The 21-kb lin-14 gene is differentially spliced to generate three lin-14 transcripts that encode protein products with variable amino-terminal regions and a constant carboxy-terminal region. The sequence of the gene revealed no protein sequence similarity to any proteins in various data bases.

Second language vowel perception.

This study explored the relation between first language (L1) and second language (L2) vowel perception using synthesized front vowel stimuli that varied in F1 and F2. Bilinguals identified vowel sounds in their L1 (Spanish) and L2 (English) on different days. Individuals’ responses provided English and Spanish F1×F2 maps of the front vowel space that could be compared to each other and to the maps of Spanish monolinguals to determine whether bilinguals have separate perceptual maps for each language, whether age at L2 acquisition or length of L2 use affects the maps, and whether L2 acquisition alters the L1 map. The results indicate that L1 and L2 maps of early L2 learners are more distinct from each other than those of late L2 learners. This finding supports the hypothesis that there is a critical age after which the sounds of a language cannot be learned authentically. No support was found for the notion that L2 acquisition affects L1 perceptual targets. An interesting peripheral finding is that bilinguals can produce vowel distinctions that they cannot perceive in isolated synthesized vowels.

Sequences involved in the control of Adenovirus L1 alternative RNA splicing

During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated.

Age of learning affects the authenticity of voice-onset time (VOT) in stop consonants produced in a second language

This study examined whether Spanish-English bilinguals are able to fully differentiate Spanish and English /t/ according to voice-onset time (VOT) if they learn English as a second language (L2) in early childhood. In experiment 1, VOT was measured in Spanish words spoken by Spanish monolinguals, in English words spoken by English monolinguals, and in Spanish and English words spoken by bilinguals who learned English either as young children or as adults. As expected, the Spanish monolinguals produced /t/ with considerably shorter VOT values than the English monolinguals. Also as expected, the late L2 learners produced English /t/ with "compromise" VOT values that were intermediate to the short-lag values observed for Spanish monolinguals and the long-lag values observed for English monolinguals. The early learners' VOT values for English /t/, on the other hand, did not differ from English monolinguals' VOT. The same pattern of results was obtained for stops in utterance-medial position and in absolute utterance-initial position. The results of experiment 1 were replicated in experiment 2, where bilingual subjects were required to produce Spanish and English utterances (sentences, phrases, words) in alteration. The results are interpreted to mean that individuals who learn an L2 in early childhood, but not those who learn an L2 later in life, are able to establish phonetic categories for sounds in the L2 that differ acoustically from corresponding sounds in the native language. It is hypothesized that the late L2 learners produced /t/ with slightly longer VOT values in English than Spanish by applying different realization rules to a single phonetic category.

Presence in infected cells of nonvirion proteins encoded by adenovirus messenger RNAs of the major late transcription regions L0 and L1

The adenovirus major late promoter functions at early and intermediate times to produce a limited set of mRNAs that appear in the cytoplasm of productively infected HeLa cells. These mRNAs may be translated in cell-free systems to produce two unrelated polypeptides of approximately 13,500 M r (L0-13.5K and L0-13.6K) and a pair of related polypeptides of approximately 55,000 M r (the L1-52K/55K proteins). Radiochemical protein sequence analysis of in vitro synthesized proteins has identified the N-terminal sequences of the L0-13.5K and L0-13.6K proteins (J. B. Lewis and C. W. Anderson 1983, Virology 127, 112–123). Additional sequence analyses confirmed the identification of the open reading frame for the L0-13.5K proteins and identified the ATG encoded by nucleotides 11,040 to 11,042 from the left end of the adenovirus genome as the initial codon of the L1-52K/55K protein. Antisera raised against synthetic peptides homologous to these three amino termini were used to demonstrate the presence of the L0-13.5K protein, the L0-13.6K protein, and the L1-52K/55K proteins in extracts of HeLa cells infected by adenovirus 2. The L0-13.5K protein was detected at early, intermediate, and late times after infection. The L0-13.6K and L1-52K/55K proteins were detected only at late times. Immunofluorescence microscopy indicated that the L0-13.6K protein is distributed around the periphery of the nucleus and along fibers running the length of the cell. Nonpermeabilized infected cells were stained by anti-L0-13.6K peptide serum at a single spot on the cell surface. Neither the L0-13.6K nor the L1-52K/55K proteins were detected in purified virus.