Discontinuous sequence change of human immunodeficiency virus (HIV) type 1 env sequences in plasma viral and lymphocyte-associated proviral populations in vivo: implications for models of HIV pathogenesis.

Sequence change in different hypervariable regions of the external membrane glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) was studied. Viral RNA associated with cell-free virus particles circulating in plasma and proviral DNA present in HIV-infected peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of two currently asymptomatic hemophiliac patients over a 5-year period. HIV sequences were amplified by polymerase chain reaction to allow analysis in the V3, V4, and V5 hypervariable regions of gp120. Rapid sequence change, consisting of regular replacements by a succession of distinct viral populations, was found in both plasma virus and PBMC provirus populations. Significant differences between the frequencies of sequence variants in DNA and RNA populations within the same sample were observed, indicating that at any one time point, the predominant plasma virus variants were antigenically distinct from viruses encoded by HIV DNA sequences in PBMCs. How these findings contribute to current models of HIV pathogenesis is discussed.     

Avian oncovirus MH2: molecular cloning of proviral DNA and structural analysis of viral RNA and protein

Viral RNA, molecularly cloned proviral DNA, and virus-specific protein of avian retrovirus MH2 were analyzed. The complexity and sequence conservation of the transformation-specific v-myc sequences of MH2 RNA were compared with those of the other members of the MC29 subgroup of acute leukemia viruses, MC29, CMII, and OK10, and with chicken cellular c-myc sequences. All T1 oligonucleotides mapping within the 1.3-kilobase coding region of MC29 v-myc have homologous counterparts in the RNAs of all MC29 subgroup viruses and in c-myc. These counterparts are either identical in composition or altered by single point mutations. Hence, the 47,000-dalton carboxy-terminal sequences of the transforming proteins of these viruses and of the cellular gene product are probably highly conserved but may contain single amino acid substitutions. T1 oligonucleotide mapping of MH2 RNA indicated that the MH2 v-myc sequences map close to the 3' end of viral RNA. A genomic library of an MH2-transformed quail cell line was prepared by using the Charon 4A vector system. By screening with an myc-specific probe, a clone containing the entire MH2 provirus (lambda MH2-1) was isolated. Digestion of cloned DNA with KpnI yielded a 5.1-kilobase fragment hybridizing to both gag- and myc-specific probes. Further restriction mapping of lambda MH2-1 DNA showed that about 1.6 kilobases of the gag gene are present near the 5' end of proviral DNA, and the conserved part of v-myc, i.e., 1.3 kilobases, is present near the 3' end of proviral DNA. These two domains are separated by a segment of at least 1 kilobase of different genetic origin, including additional unique sequences unrelated to virion genes. Tryptic peptide analysis of the gag-related protein of MH2, p100, revealed gag-specific peptides and several unique methionine-containing peptides. One of the latter is possibly shared with the polymerase precursor protein Pr180gag-pol, but no myc-specific peptides, defined for the MC29 protein p110gag-myc, appear to be present in MH2 p100. The data on viral RNA, proviral DNA, and protein of MH2 reveal a unique genetic structure for this virus of the MC29 subgroup and suggest that its v-myc gene is not expressed as a gag-related protein.     

Comparative restriction endonuclease maps of proviral DNA of the primate type C simian sarcoma-associated virus and gibbon ape leukemia virus group.

Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or point mutations or both exist throughout the entire unique portion of the genome among these viruses.     

Incorporation of excess wild-type and mutant tRNA(3Lys) into human immunodeficiency virus type 1.

Human immunodeficiency virus (HIV) particles produced in COS-7 cells transfected with HIV type 1 (HIV-1) proviral DNA contain 8 molecules of tRNA(3Lys) per 2 molecules of genomic RNA and 12 molecules of tRNA1,2Lys per 2 molecules of genomic RNA. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a human tRNA3Lys gene, there is a large increase in the amount of cytoplasmic tRNA3Lys per microgram of total cellular RNA, and the tRNA3Lys content in the virus increases from 8 to 17 molecules per 2 molecules of genomic RNA. However, the total number of tRNALys molecules per 2 molecules of genomic RNA remains constant at 20; i.e., the viral tRNA1,2Lys content decreases from 12 to 3 molecules per 2 molecules of genomic RNA. All detectable tRNA3Lys is aminoacylated in the cytoplasm of infected cells and deacylated in the virus. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a mutant amber suppressor tRNA3Lys gene (in which the anticodon is changed from TTT to CTA), there is also a large increase in the relative concentration of cytoplasmic tRNA3Lys, and the tRNA3Lys content in the virus increases from 8 to 15 molecules per 2 molecules of genomic RNA, with a decrease in viral tRNA1,2Lys from 12 to 5 molecules per 2 molecules of genomic RNA. Thus, the total number of molecules of tRNALys in the virion remains at 20. The alteration of the anticodon has little effect on the viral packaging of this mutant tRNA in spite of the fact that it no longer contains the modified base mcm 5s2U at position 34, and its ability to be aminoacylated is significantly impaired compared with that of wild-type tRNA3Lys. Viral particles which have incorporated either excess wild-type tRNA3Lys or mutant suppressor tRNA3Lys show no differences in viral infectivity compared with wild-type HIV-1.     

Phylogenetic analyses of Texas isolates indicate an evolving subtype of the clade B feline immunodeficiency viruses

Rigorous phylogenetic analyses were used to compare the nucleotide sequences of feline immunodeficiency virus strains isolated from Texas and throughout the world. The envelope V3-V4 sequences and capsid gene of the Texas isolates formed a cluster between subtypes B and E. Statistical comparisons with other published sequences confirmed that the Texas group is a unique cluster, possibly a new subtype, arising from subtype B.     

Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns.

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered in FIV vaccine and pathogenesis studies.     

Distribution of Mason-Pfizer Virus-Specific Sequences in the DNA of Primates

Iodinated Mason-Pfizer virus (MPV) 60-70S RNA has been used in molecular hybridization experiments to determine the distribution of MPV-specific proviral sequences in the DNAs of primates. Approximately 20% of the MPV genome is present as endogenous provirus in rhesus monkeys. Competitive hybridization experiments showed no homology between MPV 60-70S RNA and the 60-70S RNAs of M7, RD-114, and the simian sarcoma virus. No MPV-specific proviral sequences were detected in the DNAs of apparently normal tissues of various species of New World monkeys, apes, and humans. The part of the MPV genome that is endogenous to rhesus is also endogenous to the other species of Old World monkeys examined: baboon, African green, and patas. This was determined as a result of the following observations: (i) C(0)t(1/2) values and final extent of hybridization were the same for all four species. (ii) T(m) values of MPV 60-70S RNA and DNA of all four species were identical. (iii) The removal of MPV sequences endogenous to rhesus tissues by recycling against rhesus DNA resulted in the loss of any hybridizable MPV RNA to the DNAs of baboon, African green, and patas tissues. (iv) Mixing experiments of rhesus, African green, and baboon DNAs resulted in the same kinetics of hybridization as did rhesus DNA alone, when hybridized with MPV 60-70S RNA. These findings demonstrate that sequences that constitute an integral part of the MPV genome are conserved in the DNAs of several different species of Old World monkeys.     

Variations in integration site of avian oncornaviruses in different hosts.

We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.     

Total nucleotide sequences of the infectious cloned DNAs of bean golden mosaic virus

Complete nucleotide sequences of the infectious cloned DNA components (DNA A and B) of bean bolden mosaic virus were determined. The DNA A (2585 nucleotides) and DNA B (2647 nucleotides) have little sequence homology with each other, but both A and B contain a common region of 205 nucleotides. A possible large hairpin structure is detected in the common region. Nucleotide sequences of DNAs A and B revealed the presence of 8 potential coding regions for proteins (m.w. greater than 10,000). Among them, four open reading frames (ORFs 1-4) encode proteins of m.w. 30,000 or greater, and are individually coded in virion DNA A and B senses (+) and their complementary senses (-), respectively. The other four ORFs 5-8 are in virion DNA B(+) and its complementary sense (-). All of the ORFs 1-4 have regulatory signals for RNA synthesis (TATAA/T) in the region 5' upstream from a potential start codon ATG.     

Avian erythroblastosis virus produces two mRNA''s.

We analyzed the viral mRNA's present in fibroblast nonproducer clones transformed by avian erythroblastosis virus. Two size classes of mRNA (28 to 30S and 22 to 24S) were identified by solution hybridization with both complementary DNA strong stop and complementary DNA made against the unique sequences of avian erythroblastosis virus. Based upon the kinetics of hybridization with complementary DNA made against the unique sequences of avian erythroblastosis virus, we estimated that there were 400 to 500 copies of the 28 to 30S RNA per cell and 200 to 250 copies of the 22 to 24S RNA per cell. Both RNA species were packaged in the virion. In vitro translation of the 28 to 30S virion RNA yielded a 75,000-dalton protein which was the 75,000-dalton gag-related polyprotein found in avian erythroblastosis virus-transformed cells. In vitro translation of the 22 to 24S virion RNA yielded two proteins (46,000 and 48,000 daltons). This indicates that there may be two genes in avian erythroblastosis virus, one coding for the 75,000-dalton gag-related polyprotein and the second coding for the 46,000- or 48,000-dalton protein or both.     

Subtypes of human immunodeficiency virus type 1 and disease stage among women in Nairobi, Kenya

In sub-Saharan Africa, where the effects of human immunodeficiency virus type 1 (HIV-1) have been most devastating, there are multiple subtypes of this virus. The distribution of different subtypes within African populations is generally not linked to particular risk behaviors. Thus, Africa is an ideal setting in which to examine the diversity and mixing of viruses from different subtypes on a population basis. In this setting, it is also possible to address whether infection with a particular subtype is associated with differences in disease stage. To address these questions, we analyzed the HIV-1 subtype, plasma viral loads, and CD4 lymphocyte levels in 320 women from Nairobi, Kenya. Subtype was determined by a combination of heteroduplex mobility assays and sequence analyses of envelope genes, using geographically diverse subtype reference sequences as well as envelope sequences of known subtype from Kenya. The distribution of subtypes in this population was as follows: subtype A, 225 (70.3%); subtype D, 65 (20.5%); subtype C, 22 (6.9%); and subtype G, 1 (0.3%). Intersubtype recombinant envelope genes were detected in 2.2% of the sequences analyzed. Given that the sequences analyzed represented only a small fraction of the proviral genome, this suggests that intersubtype recombinant viral genomes may be very common in Kenya and in other parts of Africa where there are multiple subtypes. The plasma viral RNA levels were highest in women infected with subtype C virus, and women infected with subtype C virus had significantly lower CD4 lymphocyte levels than women infected with the other subtypes. Together, these data suggest that women in Kenya who are infected with subtype C viruses are at more advanced stages of immunosuppression than women infected with subtype A or D. There are at least two models to explain the data from this cross-sectional study; one is that infection with subtype C is associated with a more rapid disease progression, and the second is that subtype C represents an older epidemic in Kenya. Discriminating between these possibilities in a longitudinal study will be important for increasing our understanding of the role of specific subtypes in the transmission and pathogenesis of HIV-1.     

Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: identification of a new subtype designated subtype I.

DNA sequences encoding the C2 to V3 region of envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) were amplified by PCR from uncultured peripheral blood mononuclear cells obtained from 24 of 25 HIV-1-seropositive patients from Cyprus. By using a heteroduplex mobility assay (HMA), all amplified products were studied genetically and compared with 16 previously characterized HIV-1 strains belonging to subtypes A through F. HMA results revealed that HIV-1 gp120 sequences from 15 of our patients were of subtype B of HIV-1, whereas one isolate was of subtype C. However, gp120 sequences from eight patients had no obvious similarities to the known subtypes as defined by HMA. DNA sequencing and phylogenetic analyses of molecular clones confirmed the HMA results and placed the eight undefined HIV-1 isolates into three distinct genetic clusters. On the basis of branch topology and lengths of the phylogenetic tree, we conclude that one group consisting of three clones from two patients represents a new HIV-1 env subtype, which we have termed subtype I. The remaining two sequence clusters, consisting of five sequences from four patients and two sequences from two other patients, are distally related to subtypes A and F. These data demonstrate the extensive heterogeneity of HIV-1 in Cyprus, including the presence of new subtype.     

Identification of human immunodeficiency virus type 1 envelope genes recombinant between subtypes B and F in two epidemiologically linked individuals from Brazil.

Sequence analysis of a human immunodeficiency virus type 1 env gene PCR amplified from a Brazilian woman's peripheral blood mononuclear cell DNA (sample RJIO1) showed that it was likely to have been derived from a double recombination event between human immunodeficiency virus type 1 subtypes B and F. The major portion of the gp120 coding sequence belonged to the B lineage, but a segment of the C2 to V3 region (approximately 135 nucleotides) clearly associated with sequences of the F lineage. The subtype F-like segment had 15 noncontiguous signature nucleotides in common with Brazilian subtype F sequences that were not found, or were rare, in subtype B sequences. In contrast, this same segment had only 3 signature nucleotides shared with subtype B sequences and not present in the Brazilian subtype F sequences. Phylogenetic analysis, amino acid signature pattern analysis, and the pattern of synonymous mutations all supported the hypothesis of a recombinational origin of the RJIO1 sequence. Related recombinant genes were also detected in peripheral blood mononuclear cell DNA obtained from the woman's recent sexual partner, indicating that the recombination event probably occurred at some previous time in the chain of virus transmission. Divergent viral sequences in the V3 region were found in the male sexual partner, while a relatively homogeneous viral population was detected in the woman, consistent with her recent infection.     

Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type 1 infection in humans: evidence for viral replication in all stages of HIV disease.

Studies of cultivatable human immunodeficiency virus type 1 (HIV-1) from plasma samples from infected patients have shown a correspondence between increasing viral burden and disease progression, but these measurements are selective and thus nonrepresentative of the in vivo viral load. Quantitation of proviral DNA sequences by the polymerase chain reaction in purified CD4+ T cells has shown a similar relationship but does not provide a measure of viral gene expression. We have studied viral DNA, genomic RNA, and spliced mRNA expression of HIV-1 in infected patients with a quantitative polymerase chain reaction assay. Viral RNA expression is detected in all stages of infection. These data show that the natural history of HIV infection is associated with a shift in the balance of viral expression favoring the production of genomic RNA without a preceding period of true viral latency.     

Diversity of mammary tumor viral genes within the genus Mus, the species Mus musculus, and the strain C3H.

Proviral sequences complementary to the C3H mouse mammary tumor virus RNA genome are present in the DNA of early occurring mammary tumors of C3H/HeN mice and are absent from apparently normal C3H/HeN tissues; these sequences are non-germ line transmitted in C3H/HeN mice and have been termed tumor-associated sequences; (W. Drohan et al., J. Virol. 21:986-995, 1977). We report here that tumor-associated sequences are present in the DNA of spontaneous mammary tumors that occur early in the life of several inbred, high-tumor-incidence mouse strains but are absent in mammary tumors that occur later in life in low- and moderate-tumor-incidence strains. These sequences are also absent in apparently normal organs tested from numerous laboratory mouse strains, feral mice, Mus musculus subspecies, and other Mus species. Sequences represented in tumor-associated sequence RNA, however, are present as endogenous provirus in GR mice (at approximately four copies per haploid genome) and in two of five substrains of C3H mice tested (at approximately one copy per haploid genome). The two substrains of C3H mice positive for endogenous tumor-associated sequence provirus were recently (circa 1930) separated from the negative substrains of C3H mice. The results may be explained by the unlikely chance segregation of proviral sequences or by the recent integration of viral genes (within the last few decades). Whereas radioactively labeled mouse mammary tumor virus 60-70S RNA or complementary DNA detected mouse mammary tumor virus-related proviral information in all laboratory mouse strains, feral mice, subspecies of M. musculus, and other species of Mus, the use of tumor-associated sequence RNA clearly revealed the genetic diversity that may exist between different colonies or substrains of "inbred" laboratory mice commonly used in cancer research.     

检测HIV-1载量的荧光实时定量PCR技术的建立及其应用

准确测定HIV 1的前病毒载量和病毒载量的技术 ,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面 ,都具有十分重要的应用价值。以定量的HIV 1DNA和RNA为标准外参照 ,利用SYBRGreen荧光染料和GeneAmp5 70 0SequenceDetectionSystem (5 70 0系统 ) ,建立了测定HIV 1的前病毒载量和病毒载量的荧光实时定量PCR技术。以病毒感染细胞和培养上清为材料 ,测定了三种化合物 (AZT ,GL和WT)对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性 ,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较。根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小 ,提出以荧光实时定量PCR技术测定前病毒载量 ,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV 1前病毒方面有特别的价值。     

Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).