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PubMed1991

Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons.

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-P1 fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Identification of feline immunodeficiency virus rev gene activity.

We constructed 16 deletion mutants from an infectious molecular clone of feline immunodeficiency virus (FIV) and a reporter plasmid carrying the bacterial chloramphenicol acetyltransferase (CAT) gene to identify the rev transactivator activity of the virus. Cotransfections of various mutants and the rev reporter clone bearing a portion of FIV env in addition to the CAT gene revealed that the sequence important for the augmentation of CAT production was located in three separate parts of the virus genome. This enhancement was FIV specific in that the human retrovirus rev and rex gene products did not activate the reporter. The phenotypic properties of an FIV proviral mutant containing a small deletion in the genome were similar to those of rev mutants derived from primate immunodeficiency viruses. These results indicate that FIV, like the other lentiviruses, contains the rev gene in its genome.

Animals↗

PubMed1991

Nucleotide sequences important for translation initiation of enterovirus RNA.

An infectious cDNA clone was constructed from the genome of coxsackievirus B1 strain. A number of RNA transcripts that have mutations in the 5' noncoding region were synthesized in vitro from the modified cDNA clones and examined for their abilities to act as mRNAs in a cell-free translation system prepared from HeLa S3 cells. RNAs that lack nucleotide sequences at positions 568 to 726 and 565 to 726 were found to be less efficient and inactive mRNAs, respectively. To understand the biological significance of this region of RNA, small deletions and point mutations were introduced in the nucleotide sequence between positions 538 and 601. Except for a nucleotide substitution at 592 (U----C) within the 7-base conserved sequence, mutations introduced in the sequence downstream of position 568 did not affect much, if any, of the ability of RNA to act as mRNA. Except for a point mutation at 558 (C----U), mutations upstream of position 567 appeared to inactivate the mRNA. In the upstream region, a sequence consisting of 21 nucleotides at positions 546 to 566 is perfectly conserved in the 5' noncoding regions of enterovirus and rhinovirus genomes. These results suggest that the 7-base conserved sequence functions to maintain the efficiency of translation initiation and that the nucleotide sequence upstream of position 567, including the 21-base conserved sequence, plays essential roles in translation initiation. A deletion mutant whose genome lacks the nucleotide sequence at positions 568 to 726 showed a small-plaque phenotype and less virulence against suckling mice than the wild-type virus. Thus, reduction of the efficiency of translation initiation may result in the construction of enteroviruses with the lower-virulence phenotype.

Animals↗

PubMed1992

Identification of a feline immunodeficiency virus gene which is essential for cell-free virus infectivity.

Feline immunodeficiency virus (FIV) contains at least three small open reading frames (ORFs) in the genome, in addition to the three structural genes. Two of these ORFs (putative vif and ORF-A) have unknown functions. Northern (RNA) blot analysis of mRNAs from an FIV-infected cell line showed that the putative-vif-specific mRNA was expressed as a 5.2-kb species. To examine the function of the putative vif gene, we constructed mutants carrying a deletion in either the vif-like gene or the rev gene from an infectious molecular clone of FIV. Although the vif mutant produced virion-associated reverse transcriptase at a normal level upon transfection, cell-free virus prepared from the transfected cells could not infect feline CD4+ cells. The infectivity of the vif mutant, however, was demonstrated in a coculture of the transfected cells and feline CD4+ cells. We conclude that FIV contains the vif gene, which is structurally and functionally similar to that of the primate lentiviruses.

Base Sequence↗

PubMed1992

Mutations within the 5' nontranslated region of hepatitis A virus RNA which enhance replication in BS-C-1 cells.

Passage of human hepatitis A virus (HAV) in cell culture results in attenuation of the virus as well as progressive increases in the efficiency of virus replication in cell culture. Because the presence of identical mutations within the 5' nontranslated regions (5'NTRs) of several independently isolated cell culture-adapted HAV variants suggests that the 5'NTR may play a role in determining this change in virus host range, we constructed chimeric infectious cDNA clones in which portions of the 5'NTR of cell culture-adapted HM175/p35 virus were replaced with cDNA from either wild-type virus (HM175/wt) or a second independently isolated, but closely related cell culture-adapted virus (HM175/p16). Substitution of the complete 5'NTR of HM175/p35 with the 5'NTR of HM175/wt resulted in virus with very small replication foci in continuous African green monkey kidney (BS-C-1) cells, indicating that 5'NTR mutations in HM175/p35 virus are required for optimal growth in these cells. A chimera with the 5'NTR sequence of HM175/p16 retained the large foci of HM175/p35 virus, while the growth properties of other viruses having chimeric 5'NTR sequences indicated that mutations at bases 152 and/or 203 to 207 enhance replication in BS-C-1 cells. These findings were confirmed in one-step growth experiments, which also indicated that radioimmunofocus size is a valid measure of virus replication competence in cell culture. An additional mutation at base 687 of HM175/p16 had only a minor role in enhancing growth. In contrast to their effect in BS-C-1 cells, these 5'NTR mutations did not enhance replication in continuous fetal rhesus monkey kidney (FRhK-4) cells. Thus, mutations at bases 152 and/or 203 to 207 enhance the replication of HAV in a highly host cell-specific fashion.

Animals↗

PubMed1992

Mutations in both the 2B and 2C genes of hepatitis A virus are involved in adaptation to growth in cell culture.

Oligonucleotide-directed mutagenesis of an infectious cDNA clone of wild-type hepatitis A virus was performed to determine which mutations acquired in the nonstructural 2B and 2C genes during adaptation to growth in cell culture were effective in enhancing virus growth in vitro. Results of transfection assays demonstrated that one mutation in the 2B gene and two mutations in the 2C gene were responsible for an increased efficiency in growth, but growth enhancement required the participation of at least two of the three mutations.

Animals↗

PubMed1992

A single base deletion in the 5' noncoding region of Theiler's virus attenuates neurovirulence.

Viral chimeras have been constructed through in vitro manipulations of the infectious cDNA clones of two prototypes of Theiler's murine encephalomyelitis virus: (i) the virulent GDVII strain and (ii) the less virulent BeAn and VL strains. Previous studies have suggested that the phenotypic differences in virulence between the BeAn and GDVII strains map to both the 5' noncoding and the coat protein regions of these viral genomes. It is shown here that attenuation mapped to the 5' noncoding region is due, at least in part, to an inadvertent deletion resulting from a cloning artifact of one C nucleotide out of four between positions 876 and 879 in the BeAn sequences. The in vitro growth characteristics in BHK-21 cells, however, do not reflect the large differences in neurovirulence between chimeras that are identical except for the deleted C. Another chimera with a mutation at position 877 and a deletion at 976 is also attenuated. The wild-type sequences from the less virulent strains BeAn and VL between nucleotides 1 and 933, in an otherwise GDVII chimera, do not attenuate virulence. Sequences of the 500 nucleotides of the 5' noncoding region proximal to the translation initiation codon were obtained for nine additional Theiler's virus strains. The attenuating deletions are discussed in the context of these sequences and the proposed secondary structures for the 5' noncoding region.

Animals↗

PubMed1992

Molecular characterization of a neuropathogenic and nonerythroleukemogenic variant of Friend murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a replication-competent, ecotropic type C retrovirus that was isolated after passage of the Friend virus complex through F344 rats. Unlike viruses in the Friend virus complex, it does not cause erythroleukemia but causes a rapidly progressive hind limb paralysis when injected into newborn rats and mice. We have isolated an infectious DNA clone (clone 3d) of this virus which causes neurological disease in animals as efficiently as parental PVC-211 MuLV. The restriction map of clone 3d is very similar to that of the nonneuropathogenic, erythroleukemogenic Friend murine leukemia virus (F-MuLV), suggesting that PVC-211 MuLV is a variant of F-MuLV and that no major structural alteration was involved in its derivation. Studies with chimeric viruses between PVC-211 MuLV clone 3d and wild-type F-MuLV clone 57 indicate that at least one determinant for neuropathogenicity resides in the 2.1-kb XbaI-ClaI fragment containing the gp70 coding region of PVC-211 MuLV. Compared with nonneuropathogenic ecotropic MuLVs, the env gene of PVC-211 MuLV encodes four unique amino acids in the gp70 protein. Nucleotide sequence analysis also revealed a deletion in the U3 region of the long terminal repeat (LTR) of PVC-211 MuLV clone 3d compared with F-MuLV clone 57. In contrast to the env gene of PVC-211 MuLV, particular sequences within the U3 region of the viral LTR do not appear to be required for neuropathogenicity. However, the changes in the LTR of PVC-211 MuLV may be responsible for the failure of this virus to cause erythroleukemia, because chimeric viruses containing the U3 region of F-MuLV clone 57 were erythroleukemogenic whereas those with the U3 of PVC-211 MuLV clone 3d were not.

3T3 Cells↗

PubMed1992

Changes in both gp120 and gp41 can account for increased growth potential and expanded host range of human immunodeficiency virus type 1.

Virus derived from an infectious molecular clone of the ELI strain of human immunodeficiency virus type 1 (HIV-1) replicates well in peripheral blood mononuclear cells and in some CD4-positive cell lines but exhibits a delayed time course of infection in CEM and H9 cells and fails to infect SupT1 and U937 cells. If the virus that emerges from infected H9 cells is used to infect CEM and H9 cells, the time course of infection is accelerated and the virus is able to infect U937 and SupT1 cells. In this study, we used the technique of polymerase chain reaction-single-strand conformation polymorphism to localize changes in both the extracellular gp120 and the transmembrane gp41 components of the envelope gene associated with adaptation to growth in tissue culture cell lines. Specifically, mutations were identified both in a region of gp120 implicated in CD4 binding and in the amino-terminal portion of gp41 adjacent to the region involved in fusion. No changes were found in the V3 loop of gp120, a region previously shown to be involved in viral tropism. When these mutations were introduced into the original molecular clone, they conferred an enhanced replicative capacity on ELI. These results demonstrate that two additional determinants in the HIV-1 envelope protein influence viral tropism and growth in vitro. They also may have important implications for the generation of viruses with increased growth potential and expanded host range seen in the late stages of HIV disease.

Amino Acid Sequence↗

PubMed1992

Influence of Theiler's murine encephalomyelitis virus 5' untranslated region on translation and neurovirulence.

The DA strain of Theiler's murine encephalomyelitis virus (TMEV), a picornavirus, causes a persistent, restricted infection and demyelinating disease in mice. In contrast, the GDVII strain causes an acute, fatal, neuronal disease and is highly neurovirulent. To investigate the role of the TMEV 5' untranslated region (UTR) in translational efficiency and the TMEV subgroup differences, we tested the translational efficiency of transcripts in vitro derived from plasmids containing DA, GDVII, or DA/GDVII chimeric 5' UTRs preceding a reporter gene or the rest of the TMEV genome. We demonstrated that GDVII RNA translates more efficiently in rabbit reticulocyte lysate than DA RNA and that this enhanced translation is mediated by multiple domains in the GDVII 5' UTR as well as a region of the genome outside of the 5' UTR. We also identified a region within DA nucleotides 14 to 395 which inhibits translation of DA RNA and could contribute to the persistent, restricted DA central nervous system infection; the predicted secondary structure of the 5' UTR demonstrates a remarkable stem-loop structure within this region that is relatively unique among picornaviruses. Data from experiments involving DA/GDVII chimeric 5' UTR full-length infectious cDNA clones suggested that sequences in the 5' UTR can affect the neurovirulence phenotype but that translational efficiency is necessary but not sufficient for neurovirulence. These studies emphasize the multigenic nature of neurovirulence and the importance of translation in the regulation of picornaviral gene expression.

Animals↗

PubMed1993

A distinct African lentivirus from Sykes' monkeys.

Asymptomatic infection with simian immunodeficiency virus (SIV) has been demonstrated in African Sykes' monkeys (Cercopithecus mitis albogularis), and virus isolation confirmed infection with a novel SIV from Sykes' monkeys (SIVsyk). Macaques inoculated with SIVsyk became persistently infected but remained clinically healthy. We utilized polymerase chain reaction amplification to generate a full-length, infectious molecular clone of SIVsyk. The genome organization of SIVsyk is similar to that of the other primate lentiviruses, consisting of gag, pol, vif, vpr, tat, rev, env, and nef. A unique feature is the absence of the highly conserved NF-kappa B binding site in the long terminal repeat. SIVsyk is genetically equidistant from other primate lentiviruses. Thus, SIVsyk represents a new group that is distinct from the four previously recognized primate lentivirus groups: human immunodeficiency virus type 1 (HIV-1), SIV from sooty mangabeys (SIVsmm) and HIV-2, SIV from African green monkeys (SIVagm), and SIV from mandrills (SIVmnd). The genetic differences between SIVsyk and SIVagm, isolates derived from monkeys of the same genus, underscore the potential for other distinct SIVs which have yet to be isolated and characterized.

Africa↗

PubMed1994

Attenuated hepatitis A virus: genetic determinants of adaptation to growth in MRC-5 cells.

A live candidate hepatitis A virus vaccine, developed from the HM-175 strain and adapted to growth in primary African green monkey kidney (AGMK) cells, was adapted to growth in MRC-5 cells. The nucleotide sequence of the MRC-5 cell-adapted virus was determined and compared with the known sequence of the AGMK cell-adapted virus. Thirteen unique mutations, which occurred during passage in MRC-5 cells, were identified. Four of the unique mutations were located in a cluster in the 5' noncoding region (NC), and three of the remaining nine mutations encoded amino acid changes. Infectious chimeric cDNAs were constructed from infectious cDNA clones of the AGMK cell-adapted and wild-type HM-175 viruses and PCR-amplified cDNA segments of the MRC-5 cell-adapted virus. The viruses encoded by these plasmids were recovered after transfection of cultured cells with in vitro transcripts, and their growth phenotypes in fetal rhesus kidney 4 (FRhK-4) and MRC-5 cells were determined. The important growth-enhancing mutations could be divided into three sets. Two of these were located in the 5' NC region, and the third was located in the 2C nonstructural gene. The mutations in the 5' NC region that developed during passage in MRC-5 cells were indispensable for efficient growth in MRC-5 cells, but a combination of the two groups in the 5' NC region and one in the 2C gene were required to increase growth dramatically in MRC-5 cells.

Adaptation, Physiological↗

PubMed1994

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.

Amino Acid Sequence↗

PubMed1994

Rapid phenotypic reversion of zidovudine-resistant feline immunodeficiency virus without loss of drug-resistant reverse transcriptase.

We have selected and plaque purified zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant mutants from an infectious molecular clone of feline immunodeficiency virus (FIV). The patterns of cross-resistance and drug susceptibilities of these mutants were similar to those of the AZT-resistant FIV that we previously selected in vitro from a wild-type FIV population and to those of the most common AZT-resistant clinical isolates of human immunodeficiency virus type 1. Two AZT-resistant mutants of FIV, one selected from a normal population and one selected from the molecular clone, each reverted rapidly to an AZT-sensitive phenotype when passaged in the absence of drug. Sequence analysis of the reverse transcriptase (RT)-encoding region from the plaque-purified AZT-resistant FIV revealed a single base change at position 2939, resulting in a Glu-to-Lys substitution at amino acid 202 of the RT. Similar analyses of plaque-purified revertants showed that the phenotypic reversion was not the result of a genotypic reversion at this position and that no additional mutations existed within the RT-encoding region of the revertants. Moreover, RTs purified from the mutant and revertant were both resistant to the 5'-triphosphate of AZT. These results indicate the complexity of AZT resistance and suggest the presence of additional factors, outside the RT-encoding region, which may contribute to AZT resistance.

Animals↗

PubMed1994

Differential activities of the human immunodeficiency virus type 1-encoded Vpu protein are regulated by phosphorylation and occur in different cellular compartments.

The human immunodeficiency virus type 1 (HIV-1)-specific Vpu is an 81-amino-acid amphipathic integral membrane protein with at least two different biological functions: (i) enhancement of virus particle release from the plasma membrane of HIV-1-infected cells and (ii) degradation of the virus receptor CD4 in the endoplasmic reticulum (ER). We have previously found that Vpu is phosphorylated in infected cells at two seryl residues in positions 52 and 56 by the ubiquitous casein kinase 2. To study the role of Vpu phosphorylation on its biological activity, a mutant of the vpu gene lacking both phosphoacceptor sites was introduced into the infectious molecular clone of HIV-1, pNL4-3, as well as subgenomic Vpu expression vectors. This mutation did not affect the expression level or the stability of Vpu but had a significant effect on its biological activity in infected T cells as well as transfected HeLa cells. Despite the presence of comparable amounts of wild-type and nonphosphorylated Vpu, decay of CD4 was observed only in the presence of phosphorylated wild-type Vpu. Nonphosphorylated Vpu was unable to induce degradation of CD4 even if the proteins were artificially retained in the ER. In contrast, Vpu-mediated enhancement of virus secretion was only partially dependent on Vpu phosphorylation. Enhancement of particle release by wild-type Vpu was efficiently blocked when Vpu was artificially retained in the ER, suggesting that the two biological functions of Vpu are independent, occur at different sites within a cell, and exhibit different sensitivity to phosphorylation.

Base Sequence↗

PubMed1994

A single amino acid change determines persistence of a chimeric Theiler's virus.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype.

Acute Disease↗

PubMed1994

Large deletion mutations involving the first pyrimidine-rich tract of the 5' nontranslated RNA of human hepatitis A virus define two adjacent domains associated with distinct replication phenotypes.

The 5' nontranslated RNA (5'NTR) of the HM175 strain of human hepatitis A virus contains several pyrimidine-rich regions, the largest and most 5' of which (pY1) is an almost pure polypyrimidine tract located between nucleotides (nt) 99 and 138, which includes five tandem repeats of the sequence motif (U)UUCC(C). Previous modeling of the RNA secondary structure suggested that this region was likely to be single-stranded, but repetitive RNase V1 cleavage sites within these (U)UUCC(C) motifs indicated that pY1 possesses an ordered structure. To assess the role of this domain in replication of the virus, a series of large deletion mutations were created which involved the pY1 domain of an infectious cDNA clone. Deletion of 44 nt between nt 96 and 139, including the entire pY1 domain, did not reduce the capacity of the virus to replicate in BS-C-1 or FRhK-4 cells, as assessed by the size of replication foci in radioimmunofocus assays or by virus yields under one-step growth conditions. In contrast, viable virus could not be recovered from transfected RNAs in which the deletion was extended in a 5' direction by an additional 3 nt (delta 93-134), most likely because of the destabilization of a predicted stem-loop structure upstream of pY1. Deletion mutations extending in a 3' fashion to nt 140, 141, or 144 resulted in moderately (delta 96-140 and delta 96-141) or strongly (delta 99-144, delta 116-144, and delta 131-144) temperature-sensitive replication phenotypes. Although deletion of the pY1 domain did not by itself affect the replication phenotype of virus, the additional deletion of sequence elements within the pY1 domain (nt 99 to 130) substantially enhanced the temperature-sensitive phenotype of delta 131-144 virus. These data suggest that the (U)UUCC(C) motifs within the pY1 domain are conserved among wild-type viruses in order to serve a function required during infection in vivo but not in cell culture. In contrast, the single-stranded region located immediately downstream of pY1 (nt 140 to 144) is essential for efficient replication in cultured cells at physiological temperature. Viruses with deletion mutations involving nt 140 to 144 and viruses with large pY1 deletions but normal replication phenotypes in cell culture may have attenuation properties which could be exploited for vaccine development.

Animals↗

PubMed1995

Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation.

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.

Amino Acid Sequence↗