Identification of a potential protease-coding gene in the genomes of bovine leukemia and human T-cell leukemia viruses

The genomes of bovine leukemia and human T-cell leukemia viruses both contain an unidentified region between the gag and pol genes. These regions harbor an open reading frame that is in a different phase from the reading frames of the gag and pol genes. Based on the deduced amino acid sequences, we show here that they potentially encode a gag precursor-cleaving protease, which is known to be fused to the gag and pol products of avian and murine retroviruses, respectively. This finding raises the interesting question of the expression and evolution of retroviral genes.     

Two cis-acting signals control ribosomal frameshift between human T-cell leukemia virus type II gag and pro genes.

The open reading frame of the human T-cell leukemia virus type II pro gene is arranged at a -1 position relative to the gag gene. Synthesis of the Gag-Pro fusion polyprotein is facilitated by ribosomal frameshift into the reading frame of the pro gene. Cloning of a synthetic 41-bp oligonucleotide corresponding to the gag-pro junction within a heterologous gene (nef of human immunodeficiency virus type I) and mutation analysis revealed that two cis-acting signals, an adenosine residue stretch and a dyad symmetry sequence, flanking the UAA termination codon, are required for efficient ribosomal frameshifting between gag and pro. The stability of the stem-loop structure is crucial for frameshifting.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I.

For study of the pol gene expression of human T-cell leukemia virus type I (HTLV-I), RNA was transcribed in vitro from proviral DNA and translated in rabbit reticulocyte lysates. This cell-free translation resulted in two major translation products representing the Gag and Gag-Pro polyproteins. By contrast, the Gag-Pro-Pol polyprotein could be readily observed only when translation was performed with mutant mRNA in which the protease (pro) reading frame was aligned to gag to eliminate the frameshifting event in the gag-pro overlap. The results indicated that two independent ribosomal frameshifting events are required for expression of the HTLV-I pol gene product. Studies with mutant DNAs facilitated the characterization of the primary structure of the HTLV-I mRNA responsible for the ribosomal frameshift in the pro-pol overlap and demonstrated that the frameshift occurs at the signal sequence UUUAAAC. Direct amino acid sequencing of the transframe protein localized the site of the frameshift to the asparagine codon AAC.     

DNA sequence preferences for the anti-cancer drug mitoxanthrone and related anthraquinones revealed by DNase I footprinting

Human T-cell leukemia virus type II (HTLV-II) isolated from a T-cell variant of hairy cell leukemia contains gag, pol and env genes as well as a fourth gene termed X, which can code three major open reading frames Xa, Xb and Xc. Proteins with molecular masses of 26 kDa (p26Xb) and 24 kDa (p24Xb) encoded by the Xb open reading frame were identified with antisera directed against synthetic peptides corresponding to the N-terminal and C-terminal amino acid sequences deduced from the structure of the Xb open reading frame. More than half the Xb products were found to be located in the nuclear fraction of HTLV-II-infected cells.     

Identification of a protease gene of human T-cell leukemia virus type I (HTLV-I) and its structural comparison

We determined the nucleotide sequence of a region between the gag and pol genes of a replication-competent proviral clone of a human T-cell leukemia virus type I (HTLV-I) from MT-2 cells. This region overlapping the gag and pol genes contains an open reading frame with a different phase from others. The deduced amino acid sequences show significant homology with the known protease gene of other retroviruses, and harbors highly conserved amino acid sequences that are well conserved in other retroviral protease domains. These results indicate that this open reading frame encodes a HTLV-I protease.     

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.     

Novel Endogenous Type C Retrovirus in Baboons: Complete Sequence, Providing Evidence for Baboon Endogenous Virus gag-pol Ancestry

A complete endogenous type C viral genome has been isolated from a baboon genomic library. The provirus, Papio cynocephalus endogenous retrovirus (PcEV), is 8,572 nucleotides long, and 38 to 59 proviral copies per baboon genome are found. The PcEV provirus possesses the typical simple retroviral gene organization, including two long terminal repeats and genes encoding gag, pol, and env proteins. The open reading frames for gag-pol and env are complete but have premature stop codons or frameshift mutations. The primer binding site of PcEV is complementary to tRNAGly. The gag and pol genes of PcEV are closely related to those of the baboon endogenous virus (BaEV). The env coding region of PcEV is related to the env genes of type C retroviruses. This suggests that PcEV is one of the ancestors of BaEV contributing the type C gag-pol genome fragment to the type C/D recombinant virus BaEV. Earlier it was shown that another endogenous type D virus (simian endogenous retrovirus) provided the env gene for BaEV (A. C. van der Kuyl et al., J. Virol. 71:3666-3676, 1997).     

Nucleotide sequence of the AIDS virus, LAV

The complete 9193-nucleotide sequence of the probable causative agent of AIDS, lymphadenopathy-associated virus (LAV), has been determined. The deduced genetic structure is unique: it shows, in addition to the retroviral gag, pol, and env genes, two novel open reading frames we call Q and F. Remarkably, Q is located between pol and env and F is half-encoded by the U3 element of the LTR. These data place LAV apart from the previously characterized family of human T cell leukemia/lymphoma viruses.     

Complete nucleotide sequence and genome organization of a Drosophila transposable genetic element, 297

The complete nucleotide sequence of 297, a Drosophila copia-like transposable element, was determined and compared with those of other similar Drosophila elements and mammalian retrovirus proviruses. It was found that 297 contains three long open reading frames, comparable in sizes and locations with gag, pol, and env genes in the proviruses of replication-competent retroviruses in vertebrates. The first and second open reading frames of 297 exhibit sequence homologies to gag and pol, respectively, of Moloney murine leukaemia virus. In particular, as with 17.6, another Drosophila copia-like element, the second open reading frame of 297 was shown to be very similar in its entire organization to the retroviral pol gene and to consist of three enzymatic domains. By contrast, no appreciable homology was found between the third open reading frame of 297 and the retroviral env gene. It is also suggested that 297 and 17.6 are a peculiar pair of copia-like elements recently diverged from a common progenitor.     

Translational recoding signals between gag and pol in diverse LTR retrotransposons

Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and Pol. To assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Candida albicans, and Arabidopsis thaliana. In contrast to the retroviruses, which typically encode pol in the -1 frame relative to gag, nearly half of the retroelements surveyed encode a single gag-pol open reading frame. This was particularly true for the Ty1/copia group retroelements. Most animal Ty3/gypsy retroelements, on the other hand, encode gag and pol in separate reading frames, and likely express Pol through +1 or -1 frameshifting. Conserved sequences conforming to slippery sites that specify viral ribosomal frameshifting were identified among retroelements with pol in the -1 frame. None of the plant retroelements encoded pol in the -1 frame relative to gag; however, two closely related plant Ty3/gypsy elements encode pol in the +1 frame. Interestingly, a group of plant Ty1/copia retroelements encode pol either in a +1 frame relative to gag or in two nonoverlapping reading frames. These retroelements have a conserved stem-loop at the end of gag, and likely express pol either by a novel means of internal ribosomal entry or by a bypass mechanism.     

Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease.

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.     

Translational control of retroviruses

All replication-competent retroviruses contain three main reading frames, gag, pol and env, which are used for the synthesis of structural proteins, enzymes and envelope proteins respectively. Complex retroviruses, such as lentiviruses, also code for regulatory and accessory proteins that have essential roles in viral replication. The concerted expression of these genes ensures the efficient polypeptide production required for the assembly and release of new infectious progeny virions. Retroviral protein synthesis takes place in the cytoplasm and depends exclusively on the translational machinery of the host infected cell. Therefore, not surprisingly, retroviruses have developed RNA structures and strategies to promote robust and efficient expression of viral proteins in a competitive cellular environment.