Molecular cloning and characterization of a complete Chinese hamster provirus related to intracisternal A particle genomes.

We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.     

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.     

Metastatic rat carcinoma cells express a new retrotransposon.

Genes differentially expressed by a rat bladder carcinoma NBT-II cells and their in-vivo-selected metastatic M-NBT-II variant were analysed. Amplification and cloning of a 277-bp B sequence, exclusively expressed by the M-NBT-II cells, were performed, and this sequence was detected as a 6.7-kb RNA. This fragment shares 46-50% identities with the gag-related protein of mouse and hamster Intracisternal A Particles (IAPs). Screening of a M-NBT-II cDNA library with the B probe selected a 1671-bp sequence corresponding to the 5' end of a novel retrotransposon member of the rat IAP family. This sequence has a strong identity with the Ecker Rat IAP (ERA-IAP) except for the B portion and has an open reading frame potentially encoding a 114-amino-acid gag retrovirus-related protein. Rearrangement of this new retrotransposon could be relevant with the tumor progression in our model system since it is only expressed in the M-NBT-II in-vivo-selected carcinoma metastasis.     

Nucleotide sequences of feline sarcoma virus long terminal repeats and 5' leaders show extensive homology to those of other mammalian retroviruses.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.     

A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression

Nuclear export of incompletely spliced RNAs is a prerequisite for retroviral replication. Complex retroviruses like human immunodeficiency virus (HIV) encode a viral transport factor (Rev), which binds to its target sequence on the RNA genome and directs it into the Crm-1-mediated export pathway. Other retroviruses, like Mason-Pfizer monkey virus, contain cis-acting constitutive RNA transport elements (CTE) which achieve nuclear export of intron-containing RNA via cellular transport factors. Here, we describe the identification and characterization of a novel cis-acting orientation-dependent RNA expression element in the coding region of the murine intracisternal A-type particle (IAP) MIA14. This IAP expression element (IAPE) can functionally replace the Rev system in the expression of HIV-1 Gag proteins but functions independently of Crm-1. The presence of this element is needed for the expression of the IAP Gag proteins, indicating its biological significance. The IAPE can be functionally replaced by placing a CTE on the MIA14 RNA, further supporting its role in mRNA export. Northern blot analysis revealed that total RNA, as well as cytoplasmic RNA, was increased when the element was present. The element was mapped to a predicted stem-loop structure in the 3' part of the pol open reading frame. There was no overall homology between the IAPE and the CTE, but there was complete sequence identity between short putative single-stranded loops. Deletion of these loops from the IAPE severely reduced Rev-independent Gag expression.     

The murine endogenous retrovirus MIA14 encodes an active aspartic proteinase that is functionally similar to proteinases from D-type retroviruses

Murine intracisternal A-type particles (IAPs) are endogenous retroviruses showing sequence homologies to B/D- and avian C-type retroviruses and a gene expression strategy similar to that of D-type retroviruses. These viruses form immature particles in the endoplasmic reticulum and do not release extracellular virions, but are competent for retrotransposition within the virus-producing cell. It had been assumed that lack of polyprotein processing and maturation is due to a defect in the viral proteinase (PR), but recent experiments have shown that polyprotein processing occurs when assembly of the mouse IAP MIA14 is artificially directed to the plasma membrane. We have expressed and purified recombinant MIA14 PR and show that it undergoes N- and C-terminal autoprocessing at defined sites. Using peptide cleavage and inhibition assays and in vitro cleavage of recombinant HIV-1 and MIA14 Gag polyproteins, we show that MIA14 PR is a catalytically competent enzyme comparable in its efficiency to PRs from type D exogenous retroviruses. MIA14 PR is related to the PR of Mason-Pfizer monkey virus both functionally and with respect to its expression strategy, and is distinct from HIV-1 PR with respect to substrate specificity and catalytic efficiency. These findings reveal a functional and possibly evolutionary relationship between MIA14 and D-type retroviruses and imply that a functional PR may be relevant for intracellular retrotransposition even in the case of an endogenous retrovirus that does not produce extracellular virus.     

Nucleotide sequence of simian virus 40 DNA: structure of the middle segment of the HindII + III restriction fragment B (sixth part of the T antigen gene) and codon usage

We report here the nucleotide sequence of the simian virus 40 DNA region that lies between the EcoRII restriction endonuclease cleavage sites at map positions 0.214 and 0.281. The sequence was determined by partial chemical degradation of terminally labeled DNA fragments according to the procedure of Maxam and Gilbert. This region represents 6.7% of the SV40 genome and is located in the middle of HindII + III restriction fragment B. It is expressed as part of the early 19-S messenger RNA, which codes for the large-T antigen protein. Only one open reading frame for translation can be deduced from the message strand of the DNA and this reading frame connects in phase with the one of both neighboring fragments. This publication is the last in a series of papers about the T-antigen gene, and several properties of this gene and its product are discussed. The non-randomness of codon usage is similar to that previously discussed for the late part of the genome. Moreover, it appears that the choice of a third letter can be determined by the nature of the following codon; some codons which start with a pyrimidine are almost never preceded by an adenosine and some ANN-type codons are almost never preceded by a guanosine.     

Proteins encoded by the long terminal repeat region of mouse mammary tumor virus: identification by hybrid-selected translation.

The long terminal repeat (LTR) region of mouse mammary tumor virus (MMTV) is known to contain an open reading frame of sufficient length to code for a protein of 36,000 Mr. The coding capacity of the 3' sequences of MMTV genomic RNA has been demonstrated by in vitro translation studies, which have reported the synthesis of four related proteins: p36, p24, p21, and p18. These proteins are overlapping translation products of the same open reading frame, with the smaller ones initiating at internal methionine codons. From the predicted amino acid sequence of the LTR protein, we have selected a region likely to be antigenic, obtained a synthetic peptide of that region, and raised antiserum to the peptide. The antipeptide serum specifically immunoprecipitated all four proteins from in vitro translated genomic 3' MMTV RNA, plus an additional one of 32,000 Mr. Published sequence data of MMRV LTRs show an internal AUG codon at a position which could initiate a protein of 32,000 Mr. The three smaller in vitro translation products (p24, p21, and p18) were consistently synthesized in much greater amounts than the p36 or p32 protein. The relative amount of each in vitro synthesized protein from genomic MMTV RNA could be predicted and was in good agreement with the postulated effect of flanking nucleotides on the efficiency of the respective AUG initiation codon. Polyadenylated RNAs, isolated from various mouse tissues, were selected by hybridization to plasmid DNA containing MMTV LTR sequences immobilized on nitrocellulose. In vitro translation of hybrid-selected mRNAs isolated from BALB/c mouse lactating mammary glands and carcinogen-induced mammary tumors, followed by immunoprecipitation with antipeptide serum, revealed that only one polypeptide was synthesized by the MMTV LTR-specific mRNA, the 36,000 Mr species.     

Protein-coding potential of mouse mammary tumor virus genome RNA as examined by in vitro translation.

The protein-coding capacity of the mouse mammary tumor virus genome has been examined by in vitro translation of genome length and polyadenylated subgenomic fragments of viral RNA. Intact genome RNA of about 35S programmed synthesis of the Pr77gag, Pr110gag and Pr160gag/pol precursors seen in infected cells in vivo. Polyadenylated RNA fragments of 18 to 28S encoded products whose tryptic peptide maps resembled those of the nonglycosylated precursor to the envelope glycoproteins, confirming the gene order 5'-gag-pol-env-3'. Translation of polyadenylated RNA fragments smaller than 18S yielded a series of related proteins whose peptide maps bore no resemblance to any of the virion structural proteins. Thus, a region of the mouse mammary tumor virus genome distal to the env gene appears to have an open reading frame sufficient to encode at least 36,000 daltons of protein as of yet unknown function.     

A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3

Translation initiation on HIV genomic RNA relies on both cap and Internal Ribosome Entry Site (IRES) dependant mechanisms that are regulated throughout the cell cycle. During a unique phenomenon, the virus recruits initiation complexes through RNA structures located within Gag coding sequence, downstream of the initiation codon. We analyzed initiation complexes paused on the HIV-2 gag IRES and revealed that they contain all the canonical initiation factors except eIF4E and eIF1. We report that eIF3 and the small ribosomal subunit bind HIV RNA within gag open reading frame. We thus propose a novel two step model whereby the initial event is the formation of a ternary eIF3/40S/IRES complex. In a second step, dependent on most of the canonical initiation factors, the complex is rearranged to transfer the ribosome on the initiation codons. The absolute requirement of this large structure for HIV translation defines a new function for a coding region. Moreover, the level of information compaction within this viral genome reveals an additional level of evolutionary constraint on the coding sequence. The conservation of this IRES and its properties in rapidly evolving viruses suggest an important role in the virus life cycle and highlight an attractive new therapeutic target.     

Mutation of a termination codon affects src initiation.

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.     

Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein.

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Rev-responsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy may offer the virus several advantages, including the ability to enter a state of low or latent expression in the host.     

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.