Pol Open Reading Frames Research Articles

Characterization of human foamy virus proteins expressed by recombinant vaccinia viruses.

We report the generation of recombinant vaccinia viruses (VVs) expressing the gag, pol, bel-1, and bet open reading frames of human foamy virus (HFV), and the establishment of a transient, VV-T7 RNA polymerase-directed expression system for the HFV env gene. The correct expression of the HFV proteins was demonstrated by radioimmunoprecipitation using monospecific rabbit antisera, by analysis of the subcellular distribution (for VVgag, VVpol, VVbel-1, and VVbet), and by the ability to induce syncytium formation (for the env expression system). The HFV pol gene was successfully expressed using its own ATG start codon. Foamy viruses are regarded as retroviruses with intracytoplasmatic capsid assembly. However, when VVgag and VVpol were used to study the HFV Gag-Pol protein interaction and particle formation, no HFV capsid structures were observed in singly or doubly infected cells. In addition, no cleavage of the Pr74gag precursor molecule by the pol-encoded protease was detected in doubly infected cells. Our results indicate that foamy virus particle assembly is fundamentally different from that of other retroviruses.

Role of auxiliary proteins in retroviral morphogenesis.

Though both the oncoviruses and the lentiviruses belong to the Retroviridae family, the lentiviral subfamily is comparatively complex both from the point of view of the number of viral proteins encoded by these viruses and in the regulation of their expression. In addition to the gag, pol, and env open reading frames (ORFs) present in all retroviruses, lentiviral genomes also contain novel ORFs generally not found in prototypic retroviruses. These ORFs code for a variety of auxiliary proteins which account for the tight and often intricate regulation of gene expression observed in lentiviruses. Auxiliary protein involvement in viral replication has been particularly well characterized in the most extensively studied member of the lentiviral subfamily, the human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immune deficiency syndrome (AIDS).

Release of the Hepatitis β Virus-associated DNA Polymerase from the Viral Particle by the Proteolytic Cleavage

Previous efforts for biochemical study of the human hepatitis B virus (HBV) DNA polymerase have been limited by its tight association with viral nucleocapsids. We report here that the soluble DNA polymerase from HBV particles was obtained by low pH treatment of the viral particles followed by incubation with small amounts of subtilisin. By these treatments, the approximately 100-kDa band in the activity gel assay was gradually converted to approximately 70 kDa, which subsequently showed reverse transcriptase activity on several exogenous templates. The single approximately 70-kDa active band, which did not show any DNA polymerase activity in endogenous reaction, was eluted through DEAE-Sepharose chromatography. These results suggest that the approximately 100-kDa protein, most likely the product of HBV Pol open reading frame, is tightly associated with viral nucleocapsids, and the approximately 70-kDa protein, the proteolytic cleavage product of approximately 100-kDa enzyme, is solubilized from viral particles as an active enzyme on exogenous templates.

Nucleotide sequence and transcriptional analysis of the DNA polymerase geneof Bombyx mori nuclear polyhedrosis virus

A gene encoding a putative DNA polymerase ( pol) of Bombyx mori nuclear polyhedrosis virus (BmSNPV) was cloned and sequenced. The gene included an open reading frame (ORF) encoding a polypeptide of 988 amino acids with a predicted molecular mass of 114.65 kDa. The deduced amino acid sequence of the BmSNPV pol ORF showed an overall identity of 96 and 45% to those of the Autographa californica NPV (AcMNPV) pol ORF and the Lymantria dispar NPV poi ORF, respectively, and contained sequences conserved in a variety of eukaryotic and viral replicative DNA polymerases. The BmSNPV pol lacked a canonical TATAA element but contained a G + C-rich sequence in the transcriptional initiation region. Analyses by Northern blot hybridization, RNase protection assay, primer extension, and 3′ and 5′ RACE (rapid amplification of cDNA ends) showed that at least seven different transcripts of approximately 3.1 kb that shared a common 3′ end were expressed from the BmSNPV pol. The expression of these transcripts from BmSNPV pol was regulated differentially during virus infection. Transcription of five of the seven species initiated in the close vicinity of and within the motif 5′-GCGTGCT-3′. One transcript placed its initiation site within the motif 5′-AGAGCGT-3′ and the remaining one within the motif 5′-GGCGGTGG-3′. The motifs 5′-GCGTGCT-3′ and 5′-AGAGCGT-3′ have been identified in pol and other genes of AcMNPV as conserved sequences containing transcriptional initiation sites, whereas the motif 5′-GGCGGTGG-3′, which is arranged as a direct repeat in BmSNPV pol but not in AcMNPV pol, has not been defined as the sequence responsible for transcriptional initiation sites. The BmSNPV pol transcripts were detectable at 2 hr postinfection (p.i.), peaked at 10 hr p.i., and declined to a low level by 18 hr p.i. The expression of BmSNPV pol was not inhibited but delayed dramatically by the protein synthesis inhibitor cycloheximide upon treatment of infected cells, whereas aphidicolin, an inhibitor of DNA polymerase, inhibited BmSNPV pol transcription. These results suggest a complicated and unique mechanism for the regulation of BmSNPV pol expression.

Genome Organization of a Biologically Active Molecular Clone of the Lymphoproliferative Disease Virus of Turkeys

The lymphoproliferative disease retrovirus (LPDV) induces an acute, horizontally transmitted disease of turkeys that is often fatal. Although LPDV cannot be grown in cultured cells, it was possible to isolate molecular clones of biologically active integrated provital genomes from spleens of infected turkeys. Based upon molecular hybridization and nucleotide sequence comparisons of its pol gene, LPDV was shown to represent a distinct group of avian retroviruses most closely related to avian sarcoma-leukemia viruses. Here we report the complete nucleotide sequence of the LPDV genome as well as amino acid sequence analysis of its gag gene products. The genetic organization of LPDV is characteristic of members of the oncovirus subfamily. Further sequence comparisons of the gag gene confirmed that LPDV is most closely related to Rous sarcoma virus (RSV). However, the gag, pro, and pol open reading frames (ORFs) were in different translational phases so that the expression of their mature gene products would require the double frame-shifting mechanism utilized by simian retroviruses, mouse mammary tumor virus, and human T-cell leukemia virus. In contrast, the RSV proteinase is synthesized as part of the gag precursor. The LPDV gag gene differs from that of RSV as well as from all other retroviruses in that it encodes a unique 31,000-Da (p31) protein, located between the MA and the CA coding sequences. Four short ORFs of unknown function were present. Whether the putative products of these ORFs account for the acute nature of LPDV-induced disease remains to be determined.

Probing the mechanism of ribosomal frameshifting on viral RNAs.

Introduction Over the last few years it has been recognized that a number of eukaryotic viruses use a highly efficient, programmed ribosomal frameshift mechanism to control expression of their replicases (reviewed in [ 11). Ribosomal frameshifting is a directed change in translational reading frame that allows the production of a single protein from two (or more) overlapping genes, and was first described in 1985 as the mechanism by which the gag-pol polyprotein of the retrovirus Rous sarcoma virus is expressed from the overlapping gag and pol open reading frames (ORFs) [2]. Related frameshift signals have since been documented in an increasing number of systems, including several other retroviruses, a number of eukaryotic positive-strand RNA-containing viruses, a double-stranded RNA virus of yeast and some plant RNA viruses (reviewed in [3]). The phenomenon is not restricted to viruses; frameshift signals of the ‘retrovirus type’ have recently been found in a number of Escherichia coli insertion elements (reviewed in [4]) and in a cellular gene, the dnaX gene of E. coli [ S ] . In this paper, I will describe the ck-acting mRNA elements implicated in frameshifting, and discuss two experimental approaches I and my co-workers are taking to investigate the mechanism of the process.

Expression of HERV-K Proviruses in Human Leukocytes

HERV-K is a 50-copy, human endogenous, class 1 retroviral element that contains some polycistrons with gag, pol, and env open reading frames. Although expression of HERV-K proviruses has been shown in cultured human cell lines, expression of these elements has not been shown in human blood leukocytes. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection techniques, we show HERV-K pol gene expression in human blood leukocytes. Expression in blood leukocytes from 7 normal individuals was from a variety of different HERV-K proviruses, while restricted expression was observed in blood cells of 5 leukemia patients and 3 polycythemia vera patients. Evidence is presented suggesting that the restricted expression in leukemia blood cells is a result of gene regulation, not gene amplification.

Expression of HERV-K proviruses in human leukocytes

HERV-K is a 50-copy, human endogenous, class 1 retroviral element that contains some polycistrons with gag, pol, and env open reading frames. Although expression of HERV-K proviruses has been shown in cultured human cell lines, expression of these elements has not been shown in human blood leukocytes. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection techniques, we show HERV-K pol gene expression in human blood leukocytes. Expression in blood leukocytes from 7 normal individuals was from a variety of different HERV-K proviruses, while restricted expression was observed in blood cells of 5 leukemia patients and 3 polycythemia vera patients. Evidence is presented suggesting that the restricted expression in leukemia blood cells is a result of gene regulation, not gene amplification.

Identification of pol-Related Gene Products of Human Foamy Virus

Human foamy virus pol gene fragments were molecularly cloned into a procaryotic expression vector. The expression pattern of the cloned fragments and nucleotide sequence analysis of the 5′ pol gene region revealed that in HFV the protease (PR) is located in the pol open reading frame. Purified recombinant proteins were used to generate antibodies in rats. In immunoblot assay, using infected cells as antigen, a precursor protein with an apparent molecular mass (Mr) of 127K was identified by antibodies directed against the reverse transcriptase (RT), RNaseH, or integrase (IN) domains of pol. With concentrated virus as antigen, the RT and RNaseH antibodies recognized a protein of 80K, the IN antiserum recognized a protein of 40K, and the PR antiserum detected a protein of approximately 10K.

Expression of human endogenous retrovirus (HERV-K) in chronic myeloid leukemia.

We have previously demonstrated the presence of a reverse transcriptase-like enzyme in retroviral particles from patients with essential thrombocythemia, polycythemia vera, and chronic myelogenous leukemia. It was subsequently shown that the human genome contains 50 copies of HERV-K. HERV-K is a human endogenous class I retroviral element that contains gag, pol, and env open reading frames. Using both reverse transcriptase-polymerase chain reaction and ribonuclease protection assays, it is demonstrated that the HERV-K pol is expressed in human blood leukocytes. The data indicates that this expression is restricted in CML white cells and is the result of gene regulation.

Ribosomal frameshifting efficiency and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation

About 1.9% of ribosomes translating the gag open reading frame of the yeast L-A double-stranded RNA virus positive strand undergo a -1 frameshift and continue translating in the pol open reading frame to make a 170-kDa gag-pol fusion protein. The importance of frameshifting efficiency for viral propagation was tested in a system where the M1 (killer toxin-encoding) satellite RNA is supported by a full-length L-A cDNA clone. Either increasing or decreasing the frameshift efficiency more than twofold by alterations in the slippery site disrupted viral propagation. A threefold increase caused by a chromosomal mutation, hsh1 (high shifter), had the same effect. Substituting a +1 ribosomal frameshift site from Ty1 with the correct efficiency also allowed support of M1 propagation. The normal -1 frameshift efficiency is similar to the observed molar ratio in viral particles of the 170-kDa gag-pol protein to the 70-kDa gag gene product, the major coat protein. The results are interpreted in terms of a packaging model for L-A.

High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay.

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.

Identification and analysis of the gag-pol ribosomal frameshift site of feline immunodeficiency virus

The pol genes of retroviruses are translated as gag-pol fusion proteins by ribosomal frameshifting within the gag-pol overlap region. During the ribosomal frameshift event, the gag open reading frame is shifted -1 nt to allow in-phase reading of the pol open reading frame. A consensus frameshift signal sequence of GGGAAAC within the gag-pol overlap region of feline immunodeficiency virus (FIV) has been identified followed by a sequence that has the potential for a pseudoknot tertiary structure. Using recombinant baculoviruses in which the frameshift occurs efficiently, the consensus sequence has been shown to be the site of the frameshift event. A mutation creating a termination codon just downstream of the putative frameshift signal sequence but upstream of the potential pseudoknot structure made a shorter gag product, but did not affect the efficiency of frameshifting. A mutation creating a termination codon just upstream of the putative frameshift signal made a shorter product and essentially abrogated frameshifting. Mutations in the first stem or the second stem in the potential pseudoknot structure severely reduced the frameshifting efficiency. Mutations which altered the length between the frameshift signal and the pseudoknot structure (the so-called spacer region) also reduced the frameshift efficiency. The insertion of a palindromic sequence, which could form a hairpin structure just upstream of the frameshift signal sequence, also affected the frameshifting. These results support the view that the ribosomal frameshift event in the FIV gag-pol region involves the identified signal sequence and appears to require the precisely positioned downstream sequence and indicated pseudoknot structure for efficient frameshifting.

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites.

Hepatitis B virus (HBV) contains a particle-associated DNA polymerase/reverse transcriptase activity encoded by the P (pol) open reading frame. Due to its low abundance, the corresponding protein has so far escaped direct detection and structural analysis. As a first step to overcome these difficulties, a series of recombinant vaccinia viruses was constructed and used for the synthesis in human hepatoma cells of both the authentic full length protein and of its functional domains. Pulse chase experiments demonstrated that the P-proteins had very short half lives in striking contrast to the viral core protein expressed in parallel with the same system. No evidence was obtained for a specific proteolytic processing of the P-protein as occurring with retroviral pol gene products. Overexpression of P-protein by recombinant vaccinia viruses was then employed to develop a highly sensitive detection method based on the in vitro phosphorylation of newly introduced target sites for protein kinase A. The usefulness of this method was demonstrated in the analysis of encapsidated P-gene products that were transiently expressed from an appropriately modified HBV genome. The results obtained indicate that the P-protein acts unprocessed, at least during the initial steps of nucleocapsid assembly and reverse transcription, and that a fraction of the P-protein molecules is linked as such to the viral DNA. Direct detection of the hepadnaviral P-protein by in vitro phosphorylation should greatly facilitate future analyses on P-protein structure and function.

A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi.

A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons.

A New Member of a Family of Site-Specific Retrotransposons Is Present in the Spliced Leader RNA Genes of Trypanosoma cruzi

A new member of a family of site-specific retrotransposons is described in the New World trypanosome Trypanosoma cruzi. This element, CZAR (cruzi-associated retrotransposon), resembles two previously described retrotransposons found in the African trypanosome T. brucei gambiense and the mosquito trypanosomatid Crithidia fasciculata in specifically inserting between nucleotides 11 and 12 of the highly conserved 39-mer of the spliced leader RNA (SL-RNA) gene. CZAR is similar in overall organization to the other two SL-RNA-associated elements. It possesses two potential long open reading frames which resemble the gag and pol genes of retroviruses. In the pol open reading frame, all three elements contain similarly arranged endonuclease domains and share extensive amino acid homology in the reverse transcriptase region. All are associated with the SL-RNA gene locus and are present in low copy numbers. They do not appear to have 5' truncated versions. All three retrotransposons are otherwise quite distinct from one another, with no significant overall amino acid homology. The presence of such retroelements inserted into the identical site within SL-RNA gene sequences in at least three evolutionarily distant trypanosomatid species argues for a functional role. Because these elements appear to have a precise target site requirement for integration, we refer to them as SL siteposons.

Purification and characterization of recombinant equine infectious anemia virus reverse transcriptase

A 1.67-kb segment of the equine infectious anemia virus pol gene, encoding a 66-kDa reverse transcriptase (RT), was cloned and expressed in Escherichia coli. Recombinant RT, purified by a combination of metal chelate affinity chromatography and ion-exchange chromatography, displays both RNA-dependent DNA polymerase and RNase H activity. The affinity of purified RT for its replication primer, tRNA(3Lys) was equivalent to that observed for human immunodeficiency virus RT. Our data suggest that an additional domain between RT-RNase H and integrase on the equine infectious anemia virus pol open reading frame is not an integral component of the RT polypeptide.

Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.

Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity.

Defective retroviruses can disperse in the human genome by intracellular transposition

Using an assay for retrotransposition detection (T. Heidmann, O. Heidmann, and J. F. Nicolas, Proc. Natl. Acad. Sci. USA 85:2219-2223, 1988), we demonstrated that a defective retrovirus deleted for the gag, pol, and env open reading frames can disperse in the genome of human HeLa cells by intracellular transposition, at a frequency close to 10(-6) events per cell per generation. Transposition requires cooperation in trans for the gag and pol gene products and may be associated with the release of low amounts of noninfectious retroviruslike particles which are the hallmarks but not the intermediates of this transposition process. Similar events could account for the dispersion at high copy number of some of the human endogenous sequences related to retroviruses and for the occurrence of noninfectious retroviruslike particles in human placenta and several tumor cell lines (reviewed by E. Larsson, N. Kato, and M. Cohen, Curr. Top. Microbiol, Immunol, 148:115-132, 1989).