Gag-pol Genes Research Articles

Protease Inhibitors as Potential Therapeutic Agents for AIDS

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

Incomplete proviral genome of mouse mammary tumour virus is present on chromosome Y of NIH Swiss and some genetically related mouse strains

An incomplete proviral genome of endogenous mammary tumour virus (MMTV) was found in DNA of several strains of mice. This MMTV-related sequence was assigned to the Y chromosome since it was clearly observed in male mice only. This MMTV provirus contained a sequence related to LTR (long terminal repeat), but not to gag-pol and env genes. NFS, NIH Swiss/S, STS/A, and DD/Tbr mice have this sequence but BALB/cHeA, SHN, SLN, C57BL/6NJcl, C3H/HeNJcl and CBA/JJcl mice are negative. In the strains containing this sequence, a DNA test for the sequence makes it possible to easily distinguish the DNAs of male or female mice.

Spontaneous immortalization of mouse embryo cells: Strain differences and changes in gene expression with particular reference to retroviral gag-pol genes

We have studied the kinetics with which cultures of primary mouse embryo cells pass through the crisis period, escape their terminal differentiation (cellular senescence), and give rise to an immortal cell line. The process is strain-dependent, with cells from the outbred Swiss CD-1 mouse being considerably more adept at forming an immortal 3T3 line than cells from the inbred SWR line; Balb/c cells appeared intermediate in their behavior. The continued presence of the tumor promoter 12- O-tetradecanoylphorbol-13-acetate or the poly(ADPribose)polymerase inhibitor 3-aminobenzamide affected the kinetics but did not seem to alter the outcome. Changes in expression of various genes, including those encoding mitogen-regulated protein (proliferin), endogenous gag-pol retrovirus sequences, insulin-like growth factor II, and a variety of protooncogenes, were monitored during the process of immortalization, and although certain changes were reproducibly characteristic of cells from a given mouse strain passed according to a specific regimen, none of the observed changes were reproducibly characteristic under all conditions of immortalization. In particular, our data indicate the absence of a strict correlation between cellular immortalization and the activation of endogenous gag-pol expression. We conclude from our observations that the establishment of permanent lines from primary mouse embryo cells in serum-containing medium reflects the selection of a variant subpopulation of cells that did not preexist but rather arose in response to the specific culture conditions by a process resembling differentiation. Multiple and complex changes in gene expression occur that are affected by the culture conditions and the strain (genotype) of the mouse.

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

A Synthetic HIV-1 Protease Inhibitor with Antiviral Activity Arrests HIV-Like Particle Maturation

A synthetic peptidemimetic substrate of the human immunodeficiency virus 1 (HIV-1) protease with a nonhydrolyzable pseudodipeptidyl insert at the protease cleavage site was prepared. The peptide U-81749 inhibited recombinant HIV-1 protease in vitro (inhibition constant Ki of 70 nanomolar) and HIV-1 replication in human peripheral blood lymphocytes (inhibitory concentration IC50 of 0.1 to 1 micromolar). Moreover, 10 micromolar concentrations of U-81749 significantly inhibited proteolysis of the HIV-1 gag polyprotein (p55) to the mature viral structural proteins p24 and p17 in cells infected with a recombinant vaccinia virus expressing the HIV-1 gag-pol genes. The HIV-1 like particles released from inhibitor-treated cells contained almost exclusively p55 and other gag precursors, but not p24. Incubation of HIV-like particles recovered from drug-treated cultures in drug-free medium indicated that inhibition of p55 proteolysis was at least partially reversible, suggesting that U-81749 was present within the particles.

Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses

The ovine-caprine lentiviruses share nucleotide homology and serological properties in their gag-pol genes and gene products but constitute two distinct biological groups represented by ovine visna virus of Icelandic origin and by caprine arthritis-encephalitis and ovine progressive pneumonia viruses of U.S. origin. Two members of each group, visna 1514 and its antigenic variant LV1-1 in the first group and CAEV/CO and S93, a field isolate virus from a local arthritic sheep, in the second group, were examined in the present study in competitive-binding studies in fibroblast and macrophage cell cultures. The cultures were preinoculated with each of the four viruses and then reinoculated with either 1514 virus or CAEV/CO, labeled with [35S]methionine. Both 1514 and CAEV/CO caused homologous interference. LV1-1 and S93 viruses shared the interference patterns of 1514 and CAEV/CO, respectively. 1514 and LV1-1 did not interfere with binding of CAEV/CO. Similarly, CAEV/CO and S93 did not interfere with binding of 1514. Remarkably, certain combinations, such as S93 plus 1514, resulted in enhanced binding of the second virus. Other experiments showed that the enhancement in binding extended to enhancement in replication of the second virus. These latter data suggested that individual cells supported replication of both viruses. Further testing of this phenomenon showed that goats could be doubly infected with two noninterfering viruses, 1514 and CAEV/CO. The ability of noninterfering related lentiviruses to infect the same cell and also the same host animal may be important in the natural history of these viruses in providing ideal conditions for the development of new recombinant viruses.

Bovine Leukaemia Virus Packaging Cell Line for Retrovirus-mediated Gene Transfer

Retroviral packaging cell lines were constructed by using the gag-pol gene of spleen necrosis virus, the gag-pol gene of Moloney murine leukaemia virus and the env gene of bovine leukaemia virus. The plasmids containing the gag-pol genes and the plasmid containing the env gene were cotransfected into NIH/3T3 and D17 cells. The cells containing the helper virus constructs were tested for their ability to package replication-defective murine leukaemia and avian reticuloendotheliosis retrovirus vectors. The titre of vector virus produced by each of the retroviral packaging cell lines was about 10(2) colony-forming units per ml of medium. Tests for events that might result in intact replication-competent retroviruses showed no evidence for the generation of such viruses. The vector viruses were able to infect dog and rat cells. Bovine cells were infected only after their cocultivation with the retroviral packaging cell lines producing murine leukaemia virus vectors, perhaps as a result of a low concentration of receptors.

New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.

Molecular cloning of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a T-lymphotropic retrovirus associated with immunodeficiency and opportunistic infections in cats. The discovery of FIV provides an opportunity for the development of a small animal model for AIDS. To initiate the molecular and biological characterization of FIV, cDNA clones were synthesized and used to isolate a proviral clone of FIV. Molecular cross-hybridization analysis of FIV with five lentiviruses revealed that nucleotide-sequence similarities exist between FIV and these lentiviruses in the gag-pol genes. However, nucleotide-sequence similarities were not seen upon comparison of the FIV long terminal repeat sequence with known viral sequences. Common antigenic determinants appeared to be shared by FIV, caprine arthritis encephalitis virus, and visna virus as shown by serological cross-reactivity of rabbit antibodies to caprine arthritis encephalitis virus and visna virus with the putative FIV core protein p28. These studies demonstrated that FIV is a member of the lentivirus subfamily and is distantly related to the AIDS lentiviruses of primates. Importantly, progeny virions of our molecular clone were infectious for experimentally inoculated cats. The availability of an infectious molecular clone will make possible a detailed dissection of the molecular pathogenesis of FIV, which may facilitate the development of vaccine and therapeutic strategies for AIDS.

Expression and processing of human immunodeficiency virus type 1 gag and pol genes by cells infected with a recombinant vaccinia virus

Human cells infected with a recombinant vaccinia virus containing human immunodeficiency virus type 1 gag-pol genes produced large amounts of human immunodeficiency virus gag proteins beginning at 1 h and peaking at 48 h postinfection. We show that these polyproteins are processed accurately into mature forms and that the viral polymerase gene is encoded as a 160-kilodalton gag-pol fusion protein, most likely by translational frameshifting from the gag into the pol reading frame.

The effect of specific mutations at and around thegag-polgene junction of Moloney murine leukaemia virus

By carrying out oligonucleotide-directed mutagenesis, in vitro, on a 3.3 kb XhoI-HindIII fragment from Moloney murine leukaemia virus Mo-MuLV proviral DNA, inserted into the phagemid pTZ19R, nine separate fragments have been prepared in which mutations have been inserted at and around the gag-pol gene junction. Using these mutant fragments Mo-MuLV proviral DNA has been reassembled and cloned into pBR322. Examination of the mutant proviral DNAs in mouse culture cells indicates that a terminator codon at the gag-pol junction is essential for function, but any of the three chain terminator codons gives an active virus. Also the region of secondary structure surrounding the terminator codon must be preserved.

Posttranslational modifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing feline leukemia virus retrovirus

The envelope glycoprotein (gp70) of a molecularly cloned, replication-defective feline leukemia virus (FeLV-FAIDS clone 61C) carries determinants for induction of fatal immunodeficiency disease, whereas the gp70 of its companion replication-competent, probably parent virus (clone 61E) does not. Immunoprecipitation analysis of the extracellular glycoproteins of 61E and EECC, a replication-competent viral construct composed of the 61C env and 3' long terminal repeat fused to the 61E gag-pol genes, demonstrated that the gp70 of EECC could be distinguished from that of 61E by both feline immune serum and a murine monoclonal antibody. Molecular weights of both the envelope precursor polyprotein (gp80) and the mature extracellular glycoprotein (gp70) of 61E were smaller than the corresponding proteins from the pathogenic EECC. Both the molecular weight disparity and monoclonal antibody discrimination of the two gp80s were abolished by inhibition of envelope protein glycosylation with tunicamycin, whereas the apparent gp70 size differences were resolved by enzymatic removal of N-linked oligosaccharides. Pulse-chase studies in EECC-infected cells demonstrated that processing of gp80 to gp70 was delayed and that this retardation of envelope glycoprotein processing could be simulated in 61E-infected cells by treatment with the glucosidase inhibitor N-methyldeoxynojirimycin, a compound that causes retention of oligosaccharides in the high-mannose form. The resultant 61E gp70 then could be recognized by sera from EECC-immunized cats. The presence of a higher content of sialic acid on the apathogenic 61E gp70 indicated that oligosaccharides of 61E and EECC gp70 were processed differently. These data suggested that the unique biochemical properties which distinguish the envelope glycoproteins of the FeLV-FAIDS variant from its companion apathogenic parent virus were responsible for T-cell cytopathicity and induction of immunodeficiency disease. Further biochemical characterization of these glycoproteins should be useful in understanding the pathogenic mechanisms of immunodeficiency disease induced by retroviruses.

Expression of the human immunodeficiency virus gag gene in insert cells

Regions of the gag-pol gene of human immunodeficiency virus (HIV), the causative agent of AIDS, have been cloned into the polyhedrin gene of the baculovirus Autographs californica nuclear polyhedrosis virus. When these recombinant viruses were used to infect insect cells, the cells produced gag-related proteins which could be immunoprecipitated with serum from AIDS patients. The major proteins produced by Acgag1, which contained the entire gag gene and a small portion of the pol gene, had molecular weights of 55,000 and 40,000 Da. Acgag2, which contained a larger portion of the pol gene in addition to the gag coding sequences, produced a major protein of 24,000 Da and only minor amounts of the 55,000- and 40,000-Da proteins. The implications of these results with respect to proteolytic processing of HIV gag proteins as well as the potential diagnostic use of this system are discussed.

Natural UAG suppressor glutamine tRNA is elevated in mouse cells infected with Moloney murine leukemia virus.

Two species of glutamine tRNA were isolated from mouse liver and their nucleotide sequences were determined. The minor glutamine tRNA(tRNA(UmUGGln)) that possesses UmUG (where Um stands for 2'-O-methyluridine) as the anticodon sequence was found to have suppressor activity for the UAG termination codon of tobacco mosaic virus RNA in a rabbit reticulocyte in vitro translation system. The amount of this suppressor glutamine tRNA in mouse liver was 1-2% of the amount of the major glutamine tRNA(tRNA(CUGGln)) that has the CUG anticodon sequence, but it was markedly increased in NIH 3T3 cells infected with Moloney murine leukemia virus and in Ehrlich ascites cells. These results support the hypothesis that tRNA(UmUGGln) actually functions in vivo as a suppressor tRNA that recognizes the UAG termination codon located at the gag-pol gene junction of Moloney murine leukemia virus and results in the synthesis of the virus-encoded protease.

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.

Genetic analysis of a new subgroup of human and simian T-lymphotropic retroviruses: HTLV-IV, LAV-2, SBL-6669, and STLV-IIIAGM.

A new primate retrovirus, STLV-IIIAGM, has been recently isolated from healthy African green monkeys and is apparently nonpathogenic in its natural host. However, spontaneous infection as well as inoculation of STLV-IIIAGM into macaques induces a disease with clinical features that resemble human AIDS. Independent isolates of human retroviruses, serologically closely related to STLV-IIIAGM, have been obtained from healthy individuals (HTLV-IV) and patients with immunodeficiency (LAV-2FG and SBL 6669) from West Africa. The latter have also been referred to as HIV-2 because, like HTLV-III/HIV-1, they may be associated with immune deficiency, or as West African human retroviruses because of their prevalence and probable origin from that region. We have molecularly cloned the STLV-IIIAGM genome and have generated probes from the gag-pol and envelope genes to analyze the genetic relatedness of these simian and human retroviruses. Our results indicate that all these retroviruses are genetically closely related to each other, HTLV-IV and STLV-IIIAGM differing only by a few restriction enzyme sites while LAV-2FG and SBL 6669 exhibit greater polymorphism from HTLV-IV/STLV-IIIAGM. These data mirror the variable degree of relatedness among members of the first subgroup of human retroviruses, HTLV-III/HIV.

Glutamine starvation of murine leukaemia virus-infected cells inhibits the readthrough of the gag-pol genes and proteolytic processing of the gag polyprotein.

The production of Moloney murine leukaemia virus from chronically infected cells was inhibited after starvation of glutamine. While the rate of synthesis of the precursor of the core proteins, Pr65gag, was not affected in the starved cells, its proteolytic processing was blocked. Pulse-chase experiments indicated that glutamine was required during the synthesis of Pr65gag to facilitate its subsequent processing. In addition, the synthesis of Pr200gag-pol, the precursor of the protease, reverse transcriptase and endonuclease, was inhibited in the glutamine-starved cells. Starvation for other essential amino acids such as tyrosine and isoleucine affected neither the synthesis nor the processing of the virus proteins. These results suggest that the readthrough mechanism which enables synthesis of the Pr200gag-pol polyprotein is modulated in the chronically infected cells by glutamine levels. Since the viral protease is part of the pol gene, its synthesis may be inhibited in the glutamine-starved cells and Pr65gag is therefore not processed.

HTLV-III gag Protein Is Processed in Yeast Cells by the Virus pol -Protease

The gag-pol gene of HTLV-III (human T-lymphotropic virus), the virus linked to AIDS (acquired immune deficiency syndrome), was expressed in yeast, and processing of the gag precursor into proteins of the same size as those in the virion was observed. Processing of the gag gene in yeast cells mimics the process that naturally occurs in mammalian cells during maturation of virions. Therefore it was possible to perform mutational analysis of the virus genome to localize the gene that codes for the protease function to the amino terminal coding region of the pol gene. Since this region overlaps the gag gene, it is likely that ribosomal frameshifting occurs from gag to pol. Antibodies in all of the AIDS patients' sera tested recognized the yeast synthesized gag proteins, although the sera showed differences in relative reactivity to the individual gag proteins and the precursor. This yeast system should be valuable not only for production of viral proteins for diagnostic or vaccine purposes but also for analysis of the genetics and biochemistry of viral gene functions--parameters that are difficult to study otherwise with this virus.

Trans -Activator Gene of Human T-Lymphotropic Virus Type III (HTLV-III)

Human T-lymphotropic virus type III (HTLV-III) encodes a trans-acting factor that activates the expression of genes linked to the HTLV-III long terminal repeat. By functional mapping of complementary DNA transcripts of viral messenger RNA's the major functional domain of the gene encoding this factor was localized to a region immediately before the env gene of the virus, a region previously thought to be noncoding. This newly identified gene consists of three exons, and its transcription into messenger RNA involves two splicing events bringing together sequences from the 5' part (287 base pairs), middle (268 base pairs), and 3'part (1258 base pairs) of the HTLV-III genome. A similar messenger RNA with a truncated second exon (70 base pairs) does not encode a trans-acting function. It is proposed that this second messenger RNA is the transcript of a gene (3'-orf) located after the env gene. Messenger RNA's were also identified for the env and gag-pol genes of HTLV-III.