Major Viral Transcript Research Articles

The stable 2-kb LAT intron of herpes simplex stimulates the expression of heat shock proteins and protects cells from stress

During latency of herpes simplex virus type 1 (HSV-1), the major viral transcript that is detected is the latency-associated transcript (LAT) 2-kb intron. This intron is excised from a larger (∼10 kb) primary transcript. Infection of cells with HSV expressing LATs showed that their presence increased accumulation of Hsp70 protein specifically during cold shock. Transfection of cells with a plasmid, expressing the 2-kb LAT intron, showed increased translation compared to cells transfected with plasmids deleted in regions of the intron. Cold shock of cells expressing the intron resulted in an up-regulation of Hsp70 protein, but not Hsp70 mRNA. Furthermore, these cells showed increased cell viability. Using plasmid deletion mutants in the LAT gene sequence, we have shown that the effect requires full-length LAT intron. These findings show that a function of the stable 2-kb LAT intron is to protect cells from cold-induced stress and that this may be mediated via Hsp70.

RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant

Background & Aims Present therapy for chronic hepatitis B attains control only in limited proportions. Small interfering RNA (siRNA) offers a new tool with potential therapeutic applications for hepatitis B virus (HBV). Given the importance of sequence identity in the effectiveness of siRNA and the heterogeneity of HBV sequences among different isolates, a short hairpin RNA (shRNA)-expressing plasmid, pSuper/HBVS1, was developed to target a region conserved among major HBV genotypes and assess its effectiveness control of HBV. Methods HBV replication-competent plasmid was cotransfected with pSuper/HBVS1 to HuH-7 cells or to mice. The levels of viral proteins, RNA, and DNA were examined in transfected cells and animals. The effects of pSuper/HBVS1 on clinical isolates with genotypes B and C were also determined. Results pSuper/HBVS1 significantly decreased levels of viral proteins, RNA, and DNA for HBV genotype A in cell culture and in mice. Comparable suppressive effects were observed on clinical isolates of genotypes B and C. A clone with a silent mutation in the target region was identified from a patient with genotype C. This mutant revealed diminished sensitivity to pSuper/HBVS1 and could be selected out in the presence of pSuper/HBVS1 in cell culture. Conclusions These findings indicated that shRNA could suppress HBV expression and replication for genotypes A, B, and C, promising an advance in treatment of HBV. However, the emergence of resistant mutants in HBV quasispecies should be considered.

Human papillomavirus type 31b infection of human keratinocytes and the onset of early transcription.

Human papillomaviruses (HPVs) cause a number of human tumors and malignancies, including cervical cancers. Epithelial differentiation is required for the complete HPV life cycle and can be achieved using the organotypic (raft) culture system. The CIN-612 9E cell line maintains episomal copies of HPV type 31b (HPV31b), an HPV type associated with cervical cancers. When grown in the raft system, CIN-612 9E cells form a differentiated epithelium such that infectious virions can be synthesized. Many aspects of the later stages of the HPV31b life cycle have been investigated in CIN-612 9E raft tissues. We used a biologically contained homogenization system for efficient virion extraction from raft epithelial tissues. Purified HPV31b virions were used to infect low-passage-number human foreskin keratinocytes and a variety of epithelial cell lines. Newly synthesized, spliced HPV31b transcripts were detected by reverse transcription and PCR (RT-PCR) following HPV31b infection. HPV31b infection was most efficient and reproducible in HaCaT cells. The onset of viral transcription following infection was also investigated using RT-PCR techniques. Spliced E1(*)I,E2 RNAs were present as early as 4 h postinfection (p.i.), whereas the other major viral transcripts were detected by 8 to 10 h p.i. Furthermore, we characterized the structures and temporal expression of seven novel spliced early transcripts expressed following infection.

Differential expression of HPV types 6 and 11 in condylomas and cervical preneoplastic lesions

Tumor biopsies from exophytic and flat condylomas at different locations on the genital epithelium (10 cases) and in cervical intraepithelial neoplasia (CIN) grades 1–2 (6 cases) were analysed for HPV types 6 and 11 DNA and RNA. The presence of mRNA species which could encode the E6, E7, E1M, E2, E2C, E4, E5 and L1 proteins was determined using the RNA polymerase chain reaction (PCR) technique with primers that flank previously mapped or predicted splice sites. The state of the viral DNA in the tumor biopsies was established by Southern blot analysis. We could detect the various mRNA species in biopsies from condylomas associated with both HPV types. The size of the RNA PCR products were in agreement with the previously mapped splice sites of mRNAs recovered from an experimental condyloma induced by HPV11. The major viral transcript encoding the Eli E4 protein was detected in all the tumor biopsies. From the rare transcripts the rate of detection of mRNA species encoding the E1M, E2C proteins was the highest. In 2 of 6 CIN biopsies analysed only the major viral transcript was detected. The overall results of this study suggest that early gene products of HPV types 6 and 11 may be important in the induction of cellular proliferation and condylmatous differentiation but these possibly may not be required for the development of the HPV-associated cervical neoplasia.

Temporal aspects of major viral transcript expression in hep G2 cells transfected with cloned hepatitis B virus DNA: With emphasis on the X transcript

The major transcripts of hepatitis B virus (HBV) and the kinetics of their expression were studied in a transient expression system by transfecting partially duplicated copies of HBV genome into Hep G2 cells. By Northern blotting, six species of HBV-specific transcripts could be identified. They were the pregenomic (3.6 kb), the preS1 (2.6 kb), the preS2/S (2.2 kb), the X (0.8 kb), and two spliced (2.2 kb) RNAs, respectively. The preS2/S RNA and the spliced RNAs could be distinguished when a core gene-specific probe, which could not hybridize with the former, was used. Kinetic analysis of the expression of these RNAs revealed that the X transcript exhibited a pattern different from that of other viral transcripts. Amounts of all RNAs peaked at 24–48 hr post-transfection and then gradually declined. However, the X transcript became undetectable on Day 4 post-transfection while other viral RNAs persisted for at least 10 days. The unique expression profile of the X transcript suggested that it probably behaves as an early gene and this is consistent with its proposed role as a transactivator. Nevertheless, frameshift mutations within the X ORF had no obvious effects on the activities and temporal pattern of HBV transcription in this transient expression system.

Herpes simplex virus latency-associated transcript is a stable intron.

The latency-associated transcript (LAT) is the major viral transcript detected by in situ hybridization of mouse and human sensory ganglia latently infected with herpes simplex virus type 1. The last 750 bases of LAT are complementary to infected-cell polypeptide 0, a herpes simplex virus type 1 immediate-early gene that encodes a transactivating protein that may facilitate re-activation of the virus from the latent state. Several laboratories have shown that LAT accumulates in the nucleus and is not polyadenylylated. Recently, we showed that the promoter for LAT lies 688 bases upstream from its 5' end. We report here that LAT is actually a uniquely stable intron. Furthermore, LAT effectively inhibits transactivation of gene expression by infected-cell polypeptide 0 in transient transfection assays.

Recombination resulting in unusual features in the polyomavirus genome isolated from a murine tumor cell line

Polyomavirus-induced tumor formation in the adult natural mouse host has been investigated. Tumors were produced in nude mice with the transformation-defective mutant strain NG18 after a long latency period by apparent activation of a cryptic endogenous transforming viral function. A tumor cell line, designated ScB, was established and characterized. Cells from this morphologically distinct line were unusual in that they grew in soft agar but did not form foci. They were highly tumorigenic. They had a 3.1-kilobase major viral transcript that hybridized to probes derived from regions encoding both the T antigens and the structural proteins. ScB cells expressed polyomavirus small T antigen, a slightly altered middle T antigen, and a truncated large T antigen but no capsid proteins. Middle T antigen preserved its interactions with host proteins of 60 and 37 kilodaltons and with c-src. Analysis of cDNA and genomic clones indicated that the stable viral insert in the ScB genome contained multiple copies of the viral B-enhancer. The genome contained two intragenic inversions which created novel early- to late-strand switches. A simple model for the generation of one inversion is proposed that involves the juxtaposition of two stem-loop structures at an illegitimate recombination site; the location of the inverted segment within the integrated sequence permits use of the viral late polyadenylation signal in early-region transcripts, as confirmed by DNA sequence. A repetitive sequence may facilitate recombination at the other inversion site. Both the biological consequences of the observed rearrangements and the structure of the integrated viral DNA suggest that the recombination events are nonrandom.

Susceptibility of human cells to killing by the parvoviruses H-1 and minute virus of mice correlates with viral transcription

Human fibroblasts and epithelial cells differing in their susceptibility to killing by the autonomous parvoviruses H-1 and minute virus of mice were compared for their capacity to express viral mRNAs and proteins. The transition from a parvovirus-resistant to a parvovirus-sensitive phenotype correlated with a proportional increase in the production of the three major viral transcripts and of structural and nonstructural proteins. In contrast, cell sensitization to parvovirus could not be correlated with detectable changes in virus uptake, intracellular localization of gene products, stability of viral mRNAs, or phosphorylation of viral nonstructural polypeptides. Moreover, the H-1 virus-sensitive keratinocyte line studied did not sustain a greater level of viral DNA amplification than its resistant derivative. Therefore, the differential susceptibility of the human cells tested to parvovirus infection appears to be mainly controlled at the level of transcription of the viral genome. Parvoviral gene expression could not be elevated by increasing the input multiplicity of infection in either of the cell systems analyzed. Together, these data suggest that a cellular factor(s) regulating parvoviral transcription may be modulated by oncogenic transformation or by differentiation, as both features have been shown to affect cell susceptibility to parvoviruses.

Proviral DNA integration and transcriptional patterns of equine infectious anemia virus during persistent and cytopathic infections

The structure and integration patterns of equine infectious anemia virus (EIAV) proviral DNA and the patterns of viral transcription were examined in persistent and cytopathic infections of cultured cells. The results of Southern blot analyses indicated that, in persistently infected cells, about 30% of the EIAV provirus exists as randomly integrated DNA, while the remaining 70% is equally divided between unintegrated linear and closed circular forms. The cytopathic infection, in contrast, is characterized by levels of integrated provirus ranging from 65 to more than 90% of the total proviral DNA, depending on the extent of cytopathology exhibited by the virus strain employed. In both persistent and cytopathic infections, extensive Northern (RNA) blot analyses have revealed the presence of two major virus-specific transcripts, an 8.2-kilobase (kb) full-length genomic mRNA and a 3.5-kb single-spliced mRNA. A low-abundance 1.5-kb mRNA, presumably formed by a double-splicing event of the full-length RNA, was also detected in the cytopathic EIAV infection. The two major viral transcripts are present in approximately equal quantities in persistently infected cells, while the cytopathic infection reveals nearly a 30-fold higher level of viral transcripts in which the 3.5-kb species constitutes over 75% of the total viral mRNA. The relatively high proportion of proviral DNA integration and the simple pattern of viral transcription observed during EIAV infections appeared to be different from the generally observed patterns of predominantly unintegrated proviral DNA and multi-spliced viral mRNAs in cells infected with other lentiviruses such as visna virus or human immunodeficiency virus type 1. Moreover, the data suggested that the cytopathology of EIAV may be correlated in part with the degree of proviral DNA integration and levels of viral mRNA in infected cells, particularly that of the spliced 3.5-kb mRNA.

Mapping of two novel transcripts of bovine papillomavirus type 4.

A cDNA library was synthesized using RNA from bovine papillomavirus type 4 (BPV-4)-induced papillomas. The major viral transcript was characterized by sequencing of its cDNA, primer extension mapping and S1 nuclease protection studies. The transcript initiates at multiple sites between nucleotide (nt) 777 and nt 902, contains a splice junction between nt 1016 and nt 3376 and terminates at nt 4034, using the early polyadenylation signal at nt 4009. Sequencing of viral cDNAs and mRNA revealed deviations from the reported genomic sequence between nt 3412 and nt 3460. These resulted in a temporary frameshift, abolished the translation termination codon of the E5a open reading frame (ORF), and introduced a new termination codon in the E4 ORF. The new uninterrupted E5 ORF was found to have greater homology to the E4 ORFs of other papillomaviruses than to their E5 ORFs. The E5 ORF of BPV-4 is joined to a five codon ORF in the leader exon of the major transcript, in the same way as the E4 ORF in the major transcripts of BPV-1 and human papillomavirus type 11. On this basis, it has been redesignated E4, and the viral genomic sequence revised. Two novel transcriptional promoters of BPV-4 were defined: a putative controller of the multiple major RNA start sites around nt 870 and a minor TATA box at nt 691. In addition, minor early region transcripts were mapped which initiate between nt 3071 and nt 3152. None of these transcripts utilizes the splice site at nt 3376. These messages may express E2-encoded functions.

Cottontail rabbit papillomavirus-specific transcripts in transplantable tumors with integrated DNA

The viral DNA and viral transcripts of a Vx2 and a Vx7 transplantable carcinoma lines were analyzed. Both tumor lines contain equivalent numbers of gene copies per cell of exclusively integrated viral DNA. Integration is in head-to-tail tandem repeats, in probably three sites and at least two integration sites are common. In both tumors, about half of the viral genomes share a 90-bp deletion. Common integration sites and deletions suggest that the two tumor lines were derived from the same parent tumor. The tumors contain the same major viral transcripts of 1.3 and 2.0 kb also found in both papillomas and primary carcinomas of domestic rabbits [M. Nasseri, F. O. Wettstein, and J. G. Stevens, J. Virol. 44, 263–268 (1982)]. However, the carcinoma lines differ in their minor transcripts. Vx7 has four minor transcripts of 5.2, 3.8, 3.6, and 3.0 kb and Vx2 has two of 4.8 and 3.0 kb. The 4.8-kb transcript is relatively major and appears to be identical to a major transcript present in virus-producing tumors of cottontail rabbits [M. Nasseri and F. O. Wettstein, J. Virol. 51, 706–712 (1984)].

Physical state and transcription of the cottontail rabbit papillomavirus genome in warts and transplantable VX2 and VX7 carcinomas of domestic rabbits.

The physical state and the transcription of the genome of cottontail rabbit papillomavirus (CRPV) in non-virus-producing warts and in the VX2 and VX7 transplantable carcinomas of domestic rabbits were compared. The CRPV DNA present in VX2 and VX7 carcinomas (10 to 20 and 100 to 200 genome equivalents per diploid cell, respectively) was found to be entirely integrated into the cellular DNA, most probably as head-to-tail tandem repeats, in contrast to warts, in which viral DNA (10 to 100 copies per diploid cell) was found only as free, mainly monomeric, molecules. In the VX7 tumor, ca. 50% of the viral DNA molecules were found to be longer than one genome length, indicating that viral DNA rearrangements had occurred. A major viral transcript of 1,250 bases was detected in warts and in VX2 and VX7 carcinomas. Complementary sequences were localized within the E region, the putative transforming region inferred from the nucleotide sequence of the CRPV genome (I. Giri, O. Danos, and M. Yaniv, manuscript in preparation). Analysis of heteroduplexes formed between single-stranded CRPV DNA and polyadenylated RNAs from the VX2 tumor showed that the 1,250-base RNA resulted from the splicing of the sequences corresponding to the open reading frame E6 to those corresponding to the 3' third of E2. A second viral transcript, measuring 2,000 bases, was detected in warts and, in lesser amounts than the 1,250-base species, in VX2 carcinoma, and a 2,100-base RNA was found in VX7 carcinoma. Complementary sequences to these messengers were localized to the same part of the genome as the 1,250-base species and to a contiguous fragment situated upstream. Heteroduplex analysis showed that the 2,000-base species from VX2 carcinoma resulted from the splicing of the sequences corresponding to E6 and E7 to those corresponding to the 3' third of E2. The sequences spliced out upon the maturation of the two messengers of VX2 carcinoma correspond to E1, the two-thirds of E2, and most of E4. Additional transcripts were found in VX7 carcinoma, a major 3,100-base species transcribed from the E region, and several minor species, measuring from 2,400 bases, which all hybridize with a subgenomic fragment contained in the L region encoding the viral capsid polypeptides. This could account for the antiviral antibodies found in animals bearing the VX7 carcinoma.