Avian carcinoma virus MH2 contains a transformation-specific sequence, mht, and shares the myc sequence with MC29, CMII, and OK10 viruses.

Avian carcinoma virus MH2 has been grouped together with MC29, CMII, and OK10, because all of these viruses share a transformation-specific sequence termed myc. A 5.2-kilobase (kb) DNA provirus of MH2 has been molecularly cloned. The complete genetic structure of MH2 is 5''-delta gag(1.9-kb)-mht(1.2-kb)-myc(1.3-kb)-delta env(?) and noncoding c-region (0.2-kb)-3''. delta gag, delta env, and c are genetic elements shared with nondefective retroviruses, whereas mht is a unique, possibly MH2 transformation-specific, sequence. Hybridizations with normal chicken DNA and cloned chicken c-myc DNA indicate that the mht sequence probably derives from a normal cellular gene that is distinct from the c-myc gene. The genetic structure of MH2 suggests that the delta gag and mht sequences function as a hybrid gene that encodes the p100 putative transforming protein. The myc sequence of MH2 appears to encode a second transforming function. Therefore, it seems that MH2 contains two genes with possible oncogenic function, whereas MC29, CMII, and OK10 each carries a single hybrid delta gag-myc transforming gene. It is remarkable that, despite these fundamental differences in their primary structures and mechanisms of gene expression, MH2 and MC29 have very similar oncogenic properties.     

Nucleotide sequence of avian carcinoma virus MH2: two potential onc genes, one related to avian virus MC29 and the other related to murine sarcoma virus 3611.

The 5.2-kilobase (kb) RNA genome of avian carcinoma virus MH2 has the genetic structure 5'-delta gag (0.2 kb)- mht (1.2 kb)-myc (1.4 kb)-c (0.4 kb)-poly(A) (0.2 kb)-3'. delta gag is a partial retroviral core protein gene, mht and myc are cell-derived MH2-specific sequences, and c is the 3'-terminal retroviral vector sequence. Here we have determined the nucleotide sequence of 3.5 kb from the 3' end of delta gag to the 3' end of molecularly cloned proviral MH2 DNA, in order to elucidate the genetic structure of the virus and to compare it with other mht - and myc-containing oncogenic viruses as well as with the chicken proto-myc gene. The following results were obtained: (i) delta gag- mht forms a hybrid gene with a contiguous reading frame of 2682 nucleotides that terminates with a stop codon near the 3' end of mht . The 3' 969 nucleotides of mht up to the stop codon are 80% sequence related to the onc-specific raf sequence of murine sarcoma virus 3611 (94% homologous at the deduced amino acid level). (ii) The myc sequence is preceded by an RNA splice acceptor site shared with the cellular proto-myc gene, beyond which it is colinear up to a 3'-termination codon and 40 noncoding nucleotides with the myc sequences of avian retrovirus MC29 and chicken proto-myc. Thus, myc forms, together with a 5' retroviral exon, a second MH2-specific gene. (iii) myc is followed by the 3'-terminal c region of about 400 nucleotides, which is colinear with that of Rous sarcoma virus except for a substitution near the 5' end of the long terminal repeat. It is concluded that MH2 contains two genes with oncogenic potential, the delta gag- mht gene, which is closely related to the delta gag-raf transforming gene of MSV 3611, and the myc gene, which is related to the transforming gene of MC29. Furthermore, it may be concluded that the cellular proto-onc genes, which on sequence transduction become viral onc genes, are a small group because among the 19 known onc sequences, 5 are shared by different taxonomic groups of viruses of which the mht /raf homology is the closest determined so far.     

Mutagenesis of avian carcinoma virus MH2: only one of two potential transforming genes (delta gag-myc) transforms fibroblasts.

Avian carcinoma virus MH2 contains two potential transforming genes, delta gag-mht and delta gag-myc. Thus, MH2 may be a model for two-gene carcinogenesis in which transformation depends on two synergistic genes. Most other directly oncogenic viruses contain single, autonomous transforming (onc) genes and are models for single-gene carcinogenesis. To determine which role each potential onc gene of MH2 plays in oncogenesis, we have prepared deletion and frameshift mutants of each of the two MH2 genes by in vitro mutagenesis of cloned proviral DNA and have tested transforming function and virus production in cultured primary quail cells. We have found that mht deletion mutants and wild-type virus transform primary cells and that myc deletion and frameshift mutants do not. The morphologies of cells transformed by the mht deletion mutants and by wild-type MH2 are similar yet vary considerably. Nevertheless, typical mutant transformed cells can often be distinguished from cells transformed by wild-type MH2. We conclude that the delta gag-myc gene transforms primary cells by itself, without the second potential onc gene. This myc-related gene is the smallest that has direct transforming function. delta gag-mht is without detectable transforming function but may affect transformation by delta gag-myc. Thus, MH2 behaves like a virus with a single onc gene, although it expresses two potential onc genes, and it appears not to be a model for two-gene carcinogenesis. Further work is necessary to determine whether the delta gag-mht gene possibly enhances oncogenic function of delta gag-myc or has independent oncogenic function in animals.     

Avian proto-myc genes promoted by defective or nondefective retroviruses are single-hit transforming genes in primary cells.

Lymphomas of certain strains of chickens infected by retroviruses frequently contain recombinant transforming genes in which the promoter of the cellular proto-myc gene is replaced by that of a defective rather than an intact retrovirus. Here we ask whether the resulting hybrid genes are sufficient for tumorigenic transformation like viral myc genes. Further, we ask whether retroviruses must be defective in order to mutate proto-myc to a transforming gene or whether the defectiveness plays a transformation-independent function in tumorigenesis. For this purpose the defective provirus of proviral-proto-myc recombinants from lymphomas were repaired, or intact proviruses were recombined with proto-myc genes in vitro, and then compared to recombinant proto-myc genes with defective proviruses for transforming function in quail embryo fibroblasts. It was found that a single copy of a provirus-proto-myc recombinant gene with an intact provirus is sufficient to transform a quail embryo cell in vitro. Moreover, our analyses showed that multiple internal retroviral deletions [corrected] eliminate or inhibit provirus expression. The effect of these deletions [corrected] was detectable only because the inactive proviruses were linked to the selectable, transforming proto-myc gene marker. It is consistent with our results that proviral defectiveness of recombinant proto-myc genes is necessary in vivo for the clonal growth of a transformed cell into a tumor to escape antiviral immunity. The large discrepancy between the probabilities of provirus insertion and tumorigenesis is suggested to reflect the low probabilities of spontaneous deletion of the provirus and of rare, strain-specific defects of tumor-resistance genes of the host.     

Nucleotide sequence analysis of the chicken c-myc gene reveals homologous and unique coding regions by comparison with the transforming gene of avian myelocytomatosis virus MC29, delta gag-myc.

Myelocytomatosis virus MC29 is a defective avian retrovirus with a hybrid transforming gene (delta gag-myc) consisting of a 1,358-base pair (bp) sequence from the retroviral gag gene and a 1,568-bp sequence (v-myc) shared with a cellular locus, termed c-myc. We have subjected to sequence analysis 2,735 bp of the cloned c-myc gene, which includes the v-myc-related region of 1,568 bp, an intervening sequence of 971 bp, and unique flanking sequences of 45 bp and 195 bp at the 5' and 3' ends, respectively. Analysis of the genetic information and alignment of the c-myc sequence with the known sequence of MC29 indicates that: (i) the two myc sequences share the same reading frame, including the translational termination signal; (ii) there are nine nucleotide changes between c-myc and v-myc that correspond to seven amino acid changes; (iii) the 971-bp intervening sequence of c-myc can be defined as an intron by consensus splice signals; (iv) the unique 5' sequence of c-myc could either extend its reading frame beyond the homology with v-myc or could be an intron because its junction with the myc region of the locus is a canonical 3' splice-acceptor site; (v) the v-myc contains 10 nucleotides at its 5' end not shared with the c-myc analyzed here and also not with known gag genes, probably derived from an upstream exon; and (vi) the c-myc locus can generate a mRNA whose termination signals have been identified to be located 83 bp and 119 bp from the point of divergence between the v-myc and c-myc. We conclude that the gene of the c-myc locus of the chicken and the onc gene of MC29 share homologous myc regions and differ in unique 5' coding regions and we speculate, on this basis, that their protein products may have different functions. The hybrid onc gene of MC29 must have been generated from the c-myc gene by deletion of the 5' cellular coding sequence, followed by substitution with the 5' region of the viral gag gene.     

OK10, an avian acute leukemia virus of the MC29 subgroup with a unique genetic structure

The RNA of defective avian acute leukemia virus OK10 was isolated from a defective virus particle, released by OK10-transformed nonproducer avian fibroblasts, as a 60S complex consisting of 8.6-kilobase subunits. Oligonucleotide fingerprinting and RNA.cDNA hybridization identified two sets of sequences in OK10 RNA: group-specific sequences, which are related to all nondefective members of the avian tumor virus group, and a sequence closely related to the subgroup-specific sequences (mcv) of the myelocytomatosis virus (MC29) subgroup of avian acute leukemia viruses. Hence, OK10 is classified as a member of the MC29 subgroup of avian tumor viruses, in agreement with classification based on its oncogenic spectrum. The group-specific sequences of OK10 RNA include partial (Delta) pol and env genes, a c-region, and, unlike those of all other members of the MC29 subgroup, a complete gag gene. Oligonucleotide mapping revealed 5'-gag-Deltapol-mcv-Deltaenv-c-3' as the order of the subgroup-specific and group-specific elements of OK10 RNA. The genetic unit gag-Deltapol-mcv, measuring approximately 6.4 kilobases, codes for the nonstructural, presumably transforming, 200,000-dalton OK10-specific protein and also includes the gag gene coding for the internal virion proteins. Because gag is the only intact virion gene shared in addition to regulatory RNA sequences between OK10 and nondefective avian tumor viruses, it is concluded that the gag gene is sufficient for the formation of a defective virus particle. Comparisons among the RNAs and gene products of different viruses of the MC29 subgroup show that they share 5'-terminal gag-related and internal mcv sequences but differ from each other in intervening gag-, pol-, and mcv-related sequences. It follows that the probable transforming genes and their protein products have two essential domains, one consisting of conserved 5' gag-related and the other of 3' mcv-related sequence elements. In the light of this and previous knowledge we can now distinguish two designs among five different transforming onc genes of avian tumor viruses: onc genes with coding sequences unrelated to virion genes, like those of Rous sarcoma virus and avian myeloblastosis virus, and onc genes with coding sequences that are hybrids of virion genes and specific sequences, like those of the MC29 subgroup viruses, of avian erythroblastosis virus, and of Fujinami sarcoma virus.     

Nucleotide sequence analysis of the proviral genome of avian myelocytomatosis virus (MC29).

The nucleotide sequence of the integrated proviral genome of avian myelocytomatosis virus (MC29) coding for gag-myc protein has been determined. By comparison of this nucleotide sequence with the helper virus as well as the c-myc region, it was possible to localize the junction points between helper viral and v-myc sequences. These studies demonstrate that (i) the large terminal repeat sequence of MC29 is very similar to that of Rous sarcoma virus, (ii) the viral genome has suffered extensive deletions in the gag, pol, and env genes, (iii) the gag region can code for p19, p10, and part of p27, (iv) the recombination between viral and cellular sequences occurred in the coding region of p27 such that the open reading frame extends for an additional stretch of 1,266 base pairs, resulting in a gag-myc hybrid protein, (v) the open reading frame terminated within the v-myc region 300 bases upstream of v-myc-helper viral junction, and (vi) the v-myc helper-viral junction at the 3' end occurred in the middle of env gene, rendering it defective.     

Avian acute leukemia viruses MC29 and MH2 share specific RNA sequences: Evidence for a second class of transforming genes

The genome of the defective avian tumor virus MH2 was identified as a RNA of 5.7 kilobases by its presence in different MH2-helper virus complexes and its absence from pure helper virus, by its unique fingerprint pattern of RNase T1-resistant (T1) oligonucleotides that differed from those of two helper virus RNAs, and by its structural analogy to the RNA of MC29, another avian acute leukemia virus. Two sets of sequences were distinguished in MH2 RNA: 66% hybridized with DNA complementary to helper-independent avian tumor viruses, termed group-specific, and 34% were specific. The percentage of specific sequences is considered a minimal estimate because the MH2 RNA used was about 30% contaminated by helper virus RNA. No sequences related to the transforming src gene of avian sarcoma viruses were found in MH2. MH2 shared three large T1 oligonucleotides with MC29, two of which could also be isolated from a RNase A- and T1-resistant hybrid formed between MH2 RNA and MC29 specific cDNA. These oligonucleotides belong to a group of six that define the specific segment of MC29 RNA described previously. The group-specific sequences of MH2 and MC29 RNA shared only the two smallest out of about 20 T1 oligonucleotides associated with MH2 RNA. It is concluded that the specific sequences of MH2 and MC29 are related, and it is proposed that they are necessary for, or identical with, the onc genes of these viruses. These sequences would define a related class of transforming genes in avian tumor viruses that differs from the src genes of avian sarcoma viruses.     

The ets sequence from the transforming gene of avian erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21: both loci are transcriptionally active.

Human DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be closely related to the viral equivalent by hybridization and partial sequence analysis. The transforming gene of E26 has a tripartite origin with the structure delta gag [1.2 kilobases (kb) from the viral gag gene]-myb(0.9 kb from the chicken myb gene)-ets (1.6 kb from the chicken ets gene). Human ets DNA is located on two distinct human chromosomes. The human ets-1 locus on chromosome 11 encodes a single mRNA of 6.8 kb; the second locus, ets-2 on chromosome 21, encodes three mRNAs of 4.7, 3.2, and 2.7 kb. The ets-related sequences of human DNA on chromosomes 11 and 21 are discontiguous, except for a small overlap region encoding 14 amino acids, where 12 are conserved between these two loci. By contrast, the chicken homolog has contiguous ets-1 and ets-2 sequences and is primarily expressed in normal chicken cells as a single 7.5-kb mRNA. We conclude that the ets sequence shared by the virus, the chicken, and humans is likely to contain at least two dissociable functional domains, ets-1 and ets-2. Thus, the tripartite transforming gene of E26 includes four distinct domains that may be functionally relevant for the transforming function of the virus (delta gag, myb, ets-1, and ets-2).     

A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus.

The src genes of four natural isolates of avian sarcoma viruses differ from cellular proto-src in two genetic substitutions: the promoter of the cellular gene is replaced by a retroviral counterpart, and at least six codons from the 3' terminus are replaced by retroviral or heterologous cell-derived elements. Since virus constructs with a complete proto-src coding region failed to transform avian cells but acquired transforming function by point mutations of various codons, it has been proposed that point mutation is sufficient to convert proto-src to a transforming gene. However, promoter substitution is sufficient to convert two other proto-onc genes, proto-ras and proto-myc, to retroviral transforming genes. In view of this, we have reexamined whether promoter substitution, point mutation, or both are necessary to convert proto-src into a retroviral transforming gene. It was found that a recombinant virus (RpSV), in which the src gene of Rous sarcoma virus (RSV) was replaced by the complete coding region of proto-src, transformed quail and chicken embryo cells. The oncogene of RpSV differs from the src gene of RSV in three genetic properties: (i) it is weaker--e.g., transformed cells are flatter; (ii) it is slower--e.g., focus formation takes 9 to 12 days compared to 4 days for RSV; and (iii) its host range is narrower than that of RSV--e.g., only subsets of heterogeneous embryo cells are transformed by RpSV even after weeks or months. Replacement of the proto-src 3' terminus of RpSV by that of src from RSV generates a recombinant virus (RpvSV) that equals RSV in transforming function. It is concluded that a retroviral promoter, naturally substituted via illegitimate recombination with retroviruses, is sufficient to convert at least three proto-onc genes, src, myc, and ras, to retroviral transforming genes.     

艾滋病患者感染HIV-1病毒的gag、env和pol基因变异研究

目的通过巢式聚合酶链式反应和菌落聚合酶链式反应检测艾滋病患者感染HIV-1病毒的gag、env和pol基因变异情况,为艾滋病的临床治疗提供参考。方法采集HIV-1艾滋病患者全血标本,提取HIV-1cDNA,进行巢式PCR和菌落PCR,并测序。结果巢式PCR检测70份全血标本HIV-1病毒gag、env、pol基因,分别有50、44和59份阳性,电泳检测3种基因巢式PCR产物分别为650、650和1 100bp。挑取阳性克隆体进行菌落PCR,扩增产物大小分别为750、750和1 200bp。分析3种基因序列,有两份标本gag基因在其片段尾部第118和119位间插入一个氨基酸;env基因的V3环顶端存在6种四肽序列变异方式,即GPGR、GPGQ、GQGR、GPGA、GLGR、GPGK;pol基因在PR区的第40位发生了氨基酸突变,即由TGG变异为TAG,在其第52位同样也发生了突变,由GGT变异为AGT,而在RT区第153位发生了无义突变,即突变为终止密码子。结论本地区艾滋病患者感染HIV-1病毒的gag、env和pol基因均发生了不同程度的突变,可供治疗时参考。     

Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies.

A 41-kDa unprocessed human immunodeficiency virus 2 (HIV-2) gag precursor protein that has a deletion of a portion of the viral protease assembles as virus-like particles by budding through the cytoplasmic membrane of recombinant baculovirus-infected insect cells. We have constructed six different combinations of chimeric genes by coupling the truncated HIV-2 gag gene to the neutralizing domain (V3) or the neutralizing and the CD4 binding domains (V3+CD4BD) of gp120 env gene sequences from HIV-1 or HIV-2. The env gene sequences were inserted either into the middle of the gag gene or at the 3' terminus of the gag gene. Virus-like particles were formed by chimeric gene products only when the env gene sequences were linked to the 3' terminus of the gag gene. Insertion of env gene sequence in the middle of the gag gene resulted in high-level chimeric gene expression but without the formation of virus-like particles. Three different chimeric genes [gag gene with HIV-1 V3 (1V3), gag gene with HIV-2 V3 (2V3), and gag gene with HIV-2 V3+CD4BD (2V3+CD4BD)] formed virus-like particles that were secreted into the cell culture medium. In contrast, the HIV-1 V3+CD4BD/HIV-2 gag construct did not form virus-like particles. The chimeric gag-env particles had spherical morphology and the size was slightly larger than that of the gag particles, but the chimeric particles were similar to the mature HIV particles. Western blot analysis showed that the gag-env chimeric proteins were recognized by antibodies in HIV-positive human serum and rabbit anti-gp120 serum. Rabbit anti-gag 1V3 and anti-gag 2V3 sera reacted with authentic gp120 of HIV-1 and HIV-2, respectively, and neutralized homologous HIV infectivity. Our results show that precursor gag protein has potential as a carrier for the presentation of foreign epitopes in good immunological context. The gag protein is highly immunogenic and has the ability to carry large foreign inserts; as such, it offers an attractive approach for HIV vaccine development.     

Fujinami sarcoma virus: An avian RNA tumor virus with a unique transforming gene

The oncogenic properties and RNA of the Fujinami avian sarcoma virus (FSV) and the protein it encodes were investigated and compared to those of other avian tumor viruses with sarcomagenic properties such as Rous sarcoma virus and the acute leukemia viruses MC29 and erythroblastosis virus. Cloned stocks of FSV caused sarcomas in all chickens inoculated and were found to contain a 4.5-kilobase (kb) and an 8.5-kb RNA species. The 4.5-kb RNA was identified as the genome of defective FSV because it was absent from nondefective FSV-associated helper virus and because the titer of focus-forming units increased with the ratio of 4.5-kb to 8.5-kb RNA in virus preparations. This is, then, the smallest known tumor virus RNA with a transforming function. Comparisons with other viral RNAs, based on oligonucleotide mapping and molecular hybridization, indicated that 4.5-kb FSV RNA contains a 5' gag gene-related sequence of 1 kb, an internal specific sequence of about 3 kb that is unrelated to Rous sarcoma virus, MC29, and erythroblastosis virus, and a 3'-terminal sequence of about 0.5 kb related to the conserved C region of avian tumor viruses. The lack of some or all nucleotide sequences of the essential virion genes, gag, pol, and env, and the isolation of FSV-transformed nonproducer cell clones indicated that FSV is replication defective. A 140,000-dalton, gag-related non-structural protein was found in FSV-transformed producer and nonproducer cells and was translated in vitro from full-length FSV RNA. This protein is expected to have a transforming function both because its intracellular concentration showed a positive correlation with the percentage of transformed cells in a culture and because FSV is unlikely to code for major additional proteins since the genetic complexities of FSV RNA and the FSV protein are almost the same. It is concluded that the transforming onc gene of FSV is distinct from that of Rous sarcoma virus and other avian tumor viruses with sarcomagenic properties. Hence, multiple mechanisms exist for sarcomagenic transformation of avian cells.     

Mechanisms of infectivity and replication of HIV-1 and implications for therapy

Human immunodeficiency virus type 1 (HIV-1), a retrovirus, is the etiologic agent of AIDS. Like all retroviruses, the viral genes are carried in the viral particle in the form of single-stranded RNA. Once inside a susceptible host cell, this RNA template is reverse-transcribed by virally supplied enzyme functions into a DNA copy, which becomes integrated permanently into the host's own genetic material. The genome of HIV-1, comprising approximately 10,000 bases, is much more complex than those of classic retroviruses, encoding a minimum of six gene products in addition to the gag, pol, and env genes characteristic of all retroviruses. These genes encode regulatory functions that act at diverse points in the virus life cycle. Together, they provide HIV-1 with an exceptional ability to modulate its replication depending on its host environment. This characteristic is reflected in the different stages presented by the disease and the diverse behaviors of the virus in different types of host cells. A greater understanding of the mechanics of this regulation and the factors that influence it may someday permit therapeutic intervention in the disease process that will halt virus replication and the progression of pathology in infected individuals.