Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome.

HepG2 cells, known to support the replication and virion formation of hepatitis B virus (HBV), were transfected with a cosmid constructed to contain 12 tandem head-to-tail repeats of the HBV genome for effective HBV genome expression. We detected previously identified RNAs of 3.3, 2.3, and 2.0 kilobases (kb) that code for core antigen, large surface antigen, and middle/major surface antigen, respectively. We also detected four additional RNAs of 2.1, 1.7, 1.1, and 0.7 kb [the lengths exclude the poly(A) tail]. S1 mapping and nucleotide sequencing data showed that the 2.1-kb RNA is a spliced RNA whose 5' and 3' ends are identical to those of the 3.3-kb RNA. The results suggest that the 2.1-kb RNA codes for an altered core antigen lacking the last amino acid, cysteine, and that expression of the 3.3-kb pregenomic RNA is regulated, at least in part, by splicing. The map positions of the 1.7- and 1.1-kb RNAs suggest that they code for the carboxyl-terminal portions of the putative polymerase, whereas the 0.7-kb RNA codes for the X protein.     

Isolation of novel human genomic DNA clones related to human interferon-beta 1 cDNA.

Southern blot-hybridization analyses of human DNA (from Namalwa lymphoblastoid cells) digested with the restriction endonuclease EcoRI were carried out under optimal conditions with two human fibroblast interferon (IFN-beta 1) cDNA probes, pD19 and pD24, which contain IFN-beta 1 inserts 0.8 and 0.7 kilobase (kb) long, respectively. The analyses revealed the presence of several hybridizable DNA fragments, including two of lengths 6.8 and 5.5 kb, in addition to the classical IFN-beta 1 genomic DNA fragment of length approximately equal to 2.0 kb. We have screened a human DNA library in lambda bacteriophage Charon 4A by using a 32P-labeled IFN-beta 1 insert cDNA (pD24) and thereby isolated six strongly positive human genomic DNA clones. One of these (lambda B37) represents the classical human IFN-beta 1 gene; another (lambda B37) contains a 6.8-kb EcoRI DNA fragment(s) which cross-hybridizes with the IFN-beta 1 cDNA insert probes pD19 and pD24; and the remaining four (which are identical to each other and are exemplified by lambda B4) contain two EcoRI DNA fragments approximately 5.5 and 9 kb long which also cross-hybridize the IFN-beta 1 cDNA probes. A mRNA 0.9 kb long derived from the classical IFN-beta1 gene is expressed in poly(I) . poly(C)-induced human diploid fibroblasts (FS-4 strain). Induced FS-4 cells also contain polyadenylylated RNA 1.8, 3, 5, and approximately equal to 8 kb long derived from the lambda B3 gene, all of which appear to code for biologically active human IFN-beta as tested by using the Xenopus laevis oocyte translation assay. These data strongly indicate that lambda B3 represents a novel functional IFN-beta gene. A 12-kb polyadenylylated RNA, derived from lambda B4, is expressed constitutively at a low level in FS-4 cells, but the amount of this RNA increases 5-7 hr after exposure of the cells to poly(I) . poly(C).     

Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus

Primate cells harboring the Epstein-Barr virus (EBV) genome synthesize large amounts of two small RNAs:EBER 1 and EBER 2 (EBV-encoded RNA). These RNAs are approximately 180 nucleotides long, possess 5' pppA termini, and lack poly(A). They have different T1 and pancreatic RNase digestion fingerprints. They are not found in normal B lymphocytes, in transformed B lymphocytes that lack EBV DNA, in T lymphocytes transformed by Herpesvirus ateles, or in a variety of other nonlymphoid mammalian cells. Hybridization analyses indicate that EBER 1 and EBER 2 are encoded by the EcoRI-J fragment of EBV (B95-8) DNA. In vivo both RNAs are associated with protein(s), allowing their specific precipitation by the systemic lupus erythematosus-associated antibody anti-La. The La antigen in uninfected mammalian cells consists of a heterogeneous class of small ribonucleoprotein particles, some of whose RNA components exhibit sequence homology with a highly repetitive, interspersed class of human DNA designated the Alu family. Possible functions for EBER 1 and EBER 2 in infection and cell transformation by EBV and their potential relationship to the pathogenesis of systemic lupus erythematosus are discussed.     

The major 5' end of HIV type 1 RNA corresponds to G456

In the course of studies on HIV-1 RNA structure, we determined that the main 5' end of viral RNA from virions and virus producer cells corresponds to G456 in the proviral DNA sequence, one or two nucleotides down-stream from the reported ends that correspond to G454 and G455. We mapped 5' ends using the highly accurate RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method. The reactivity of the 5' ends indicates that they are mainly capped, although the presence of some uncapped (5'-triphosphorylated) RNA cannot formally be excluded. When we used a 5' mapping method susceptible to incorporating a cytosine at the 3' end of cDNA first strands, at a position templated by the 7-methylguanosine cap, 50% of clones derived from virion RNA had incorporated the additional cytosine. Reassignment of the 5' end has consequences for the design of short RNAs used to study HIV-1 RNA structural dynamics.     

Polyadenylylated nuclear RNA encoded by Kaposi sarcoma-associated herpesvirus.

A newly recognized gamma herpesvirus known as Kaposi sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is present in Kaposi sarcomas and body-cavity-based lymphomas. Here we identify a novel abundant 1.2-kb RNA, polyadenylated nuclear RNA (PAN RNA), encoded by the virus. The majority of cDNAs produced from poly(A)-selected RNA isolated from a human body cavity lymphoma cell line 48 hr after butyrate induction of KSHV lytic replication represented PAN RNA. Within PAN RNA were two 9 and 16 nt stretches with 89% and 94% identity to U1 RNA. A third stretch of 14 nt was 93% complementary to U1. The 5' upstream region of PAN RNA contained both proximal and distal sequence elements characteristic of regulatory regions of U snRNAs, whereas the 3' end was polyadenylylated. PAN RNA was transcribed by RNA polymerase II, lacked a trimethylguanosine cap, and did not associate with polyribosomes. PAN RNA formed a speckled pattern in the nucleus typical of U snRNAs and colocalized with Sm protein. Therefore, PAN represents a new type of RNA, possessing features of both U snRNA and mRNA.