Levels Of Reverse Transcriptase Activity Research Articles

Virion-incorporated alpha-enolase suppresses the early stage of HIV-1 reverse transcription

Human immunodeficiency virus type-1 (HIV-1) particles contain not only viral-encoded but also host-encoded proteins. Interestingly, several studies showed that host proteins play a critical role in viral infectivity, replication and/or immunoreactivity in the next target cells. Here, we show that alpha-enolase (ENO1) is incorporated into HIV-1 virions and the virion-incorporated ENO1 prevents the early stage of HIV-1 reverse transcription. We found that viral particles contain two isoforms of ENO1 with different isoelectric points by two-dimensional electrophoresis. Suppression of ENO1 expression by RNA interference in the HIV-1 producer cells decreased ENO1 incorporation into virions without altering the packaging of viral structural proteins and viral production but increased viral infectivity. Although the low-level-ENO1-packaging virus maintained comparable levels of reverse transcriptase activity, viral genomic RNA and tRNALys3 packaging to the control virus, its levels of early cDNA products of reverse transcription were higher than those of the control virus. In contrast, the high-level-ENO1-packaging virus, which was produced from ENO1-overexpressing cells, showed decreased infectivity and the levels of early cDNA products. Taken together, these findings reveal a novel function of ENO1 as a negative regulation factor targeting HIV-1 reverse transcription.

Accelerated Evolution of Fetuin Family Proteins inProtobothrops flavoviridis(Habu Snake) Serum and the Discovery of an L1-Like Genomic Element in the Intronic Sequence of a Fetuin-Encoding Gene

Habu serum factor (HSF) and HSF-like protein (HLP) are fetuin family proteins isolated from Protobothrops flavoviridis (habu snake) serum with different physiological activities. A comparison of their cDNAs and intronic sequences revealed that nucleotide substitutions were primarily in protein-coding regions, and the substitution patterns indicated accelerated evolution of these proteins. Genomic DNA fragment analysis, including intron 1, revealed a 6.6-kb insertion homologous to the full-length mammalian LINE1 (L1) retrotransposable element (PfL1) only in the HLP gene. This segment retains an open reading frame (ORF) that encodes a reverse transcriptase (RT)-like protein (PfRT). We further found that a large number of homologous segments have dispersed in the habu snake genome, although we could not determine the enzymatic activities of their products. Moreover, an analysis of habu snake liver RNA indicated active transcription of the PfRT genes, suggesting that high levels of RT activity in this snake have driven the evolution of unique phenotypes of venom enzymes and serum inhibitors of them.

Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia

Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased ( p < 0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

Murine leukemia following irradiation conditioning for transplantation of lentivirally‐modified hematopoietic stem cells

Emerging reports are conclusively demonstrating the mutagenic risks involved in using retroviral vectors for gene therapy. Animal studies, as well as cases from a human clinical trial, have proven the potential of insertional leukemogenesis caused by a retroviral vector. Here, we report the observation of six T-lymphoblastic leukemia cases arising during the course of a gene therapy study for hemophilia B after transplantation of ex vivo transduced hematopoietic stem cells (HSCs) by a lentivirus vector. Three of these animals comprised secondary recipients of the same donor and LAM-PCR was performed to identify the vector integration loci. We located integrations in repeat elements of known genes, including a candidate brain-tumor locus, but none of these clones could be tracked in the leukemic blasts. Although transduced clones with an intact proviral cassette were detected in the spleen of the leukemic animals, they comprised a very small proportion, not correlating to the levels of leukemic blasts. After propagation of the latter in NOD/SCID mice, we could no longer detect the proviral cassette suggesting that the leukemic blasts were untransduced. We did, however, detect increased levels of reverse transcriptase activity in the leukemic blasts which may suggest activation of endogenous retroviruses. This study demonstrates that tumors arising in these type of gene therapy protocols are not necessarily due to vector insertional mutagenesis and highlights the importance of careful functional studies to delineate the nature of tumorigenesis.

Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

Control of telomere elongation and telomeric silencing in Drosophila melanogaster.

Chromosome length in Drosophila is maintained by the targeted transposition of two families of non-LTR retrotransposons, HeT-A and TART. Although the rate of transposition to telomeres is sufficient to counterbalance loss from the chromosome ends due to incomplete DNA replication, transposition as a mechanism for elongating chromosome ends raises the possibility of damaged or deleted telomeres, because of its stochastic nature. Recent evidence suggests that HeT-A transposition is controlled at the levels of transcription and reverse transcription. HeT-A transcription is found primarily in mitotically active cells, and transcription of a w+ reporter gene inserted into the 2L telomere increases when the homologous telomere is partially or completely deleted. The terminal HeT-A array may be important as a positive regulator of this activity in cis, and the subterminal satellite appears to be an important negative regulator in cis. A third chromosome modifier has been identified that increases the level of reverse transcriptase activity on a HeT-A RNA template and greatly increases the transposition of HeT-A. Thus, the host appears to play a role in transposition of these elements. Taken together, these results suggest that control of HeT-A transposition is more complex than previously thought.

Point mutations in the avian sarcoma/leukosis virus 3' untranslated region result in a packaging defect.

The 3' untranslated region (3' UTR) between the 3' end of env and the long terminal repeat is well conserved among avian retroviruses and is essential for efficient replication. Deletion of the dr1 element within the 3' UTR has been reported to have various effects, including reduced levels of unspliced RNA in the cytoplasm, decreased stability of unspliced RNA, decreased particle production, and decreased genomic RNA packaging. To probe the role of specific sequences within dr1 in virus replication, site-directed mutagenesis was utilized to perturb parts of the predicted secondary structure of dr1. Seven of thirteen mutations had no significant effect; the others resulted in an approximately 10- to 20-fold reduction in replication. These mutants were further characterized and found to impair cytoplasmic accumulation of unspliced RNA only slightly. Furthermore, no decreases were observed in the stability of the unspliced RNA or in the production of virus particles. Genomic RNA packaging, however, was reduced by about 10-fold. Similar amounts of particles were produced by cells containing the mutant and wild-type DNA, and all particles contained similar levels of reverse transcriptase activity. The results suggest that the region of the dr1 disrupted by the mutations plays a role in genomic RNA packaging, although that packaging may not be the only role for dr1.

Characterization of an integrase mutant of feline immunodeficiency virus.

The role of the integrase region of feline immunodeficiency virus (FIV) in viral replication was examined using an integrase mutant clone of FIV which carries a frameshift mutation in the region. Upon transfection, although the integrase mutant was able to release virus-like particles into the supernatant from the transfected cells, the virions produced by the mutant contained unprocessed gag precursor protein and undetectable levels of reverse transcriptase activity. Furthermore, the mutant virions were unable to direct the synthesis of viral DNA after infection in target cells. To understand this phenotype of the integrase mutant in more detail, we constructed a gag-pol expression plasmid from an FIV molecular clone and assayed roles of the integrase region on virus particle formation following transfection. When an inframe deletion was introduced into the protease region of the expression plasmid, the mutant was able to efficiently release gag- and gag-pol precursor proteins into the supernatant from the transfected cells. An expression plasmid with mutations in both the protease and integrase regions, however, failed to release the gag-pol precursor protein from the cells. These results suggested an essential role for the integrase region for efficient incorporation of the gag-pol precursor into the virions.

Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection

The viral integrase (IN) protein is the only viral protein known to be required for integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host cell DNA, a step in the viral life cycle that is essential for viral replication. To better understand the relationship between in vitro IN activity and IN-mediated integration of viral DNA in an infected cell, we characterized the effects of 13 IN mutations on viral replication in cultured cells. Using HIV-1 genomes that express the hygromycin resistance gene and do not express the HIV-1 env gene, we generated stocks of pseudotype virus coated with the murine leukemia virus amphotropic envelope glycoprotein, containing either wild-type or mutant HIV-1 IN. All mutants produced normal amounts of physical particles, as measured by reverse transcriptase activity and capsid protein (p24) concentration, but they formed three groups based on infectious titer and synthesis of viral DNA. Changes at the three highly conserved acidic residues in the IN core domain (D-64, D-116, and E-152) impair provirus formation without affecting viral DNA synthesis or the accumulation of viral DNA in the nucleus of the infected cell, a phenotype predicted by each mutant's lack of in vitro integrase activity. Mutations at positions N-120, R-199, and W-235 minimally affect in vitro integrase activity, but infectious titers are severely reduced, despite normal synthesis of viral DNA, implying a defect during integration in vivo. Mutations in the zinc binding region (H12C, H16V, and H16C), S81R, and a deletion of residues 32 through 275 yield noninfectious particles that synthesize little or no viral DNA following infection, despite wild-type levels of reverse transcriptase activity and viral RNA in the particles. The two latter classes of mutants suggest that IN can affect DNA synthesis or integration during infection in ways that are not appreciated from currently used assays in vitro.

Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain.

Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of reverse transcriptase activity. These data indicate that in addition to integration, IN may have an effect on the proper course of events in the viral life cycle that precede integration.

Evidence that a cell cycle regulator, E2F1, down-regulates transcriptional activity of the human immunodeficiency virus type 1 promoter

Proliferation of eukaryotic cells is orchestrated by a series of cellular proteins which participate in various stages of the cell cycle to guide the cell through mitosis. Some of these proteins, including E2F1, play a critical role in G1 and S phases by coordinately regulating expression of several important cell cycle-associated genes. On the basis of recent observations indicating a block in human immunodeficiency virus type 1 (HIV-1) replication in cells arrested in G1/S phase of the cell cycle, we sought to evaluate the regulatory action of E2F1 on transcription from the HIV-1 long terminal repeat (LTR). Results from transient transfection of cells with an E2F1 expression plasmid indicated that E2F1 has the ability to suppress basal transcriptional activity of the LTR and to diminish the extent of the Tat-induced activation of the viral promoter. Deletion analysis of the HIV-1 LTR in transfection studies revealed the presence of two major elements responsive to E2F1 repression located distally (-454 to -381) and proximally (-117 to -80) with respect to the +1 transcription start site. E2F1-mediated suppression of LTR activity was observed in a wide range of human cell lines. Expression of E2F1 by a transgene showed an inhibitory effect on the levels of reverse transcriptase activity obtained upon introduction of the proviral genome into cells. The data presented in this study suggest that cellular regulatory proteins involved in the progression of cells through the mitotic cycle could play crucial roles in determining the efficiency of HIV-1 replication during the various stages of infection. The possible roles of these factors in viral latency and activation are discussed.

Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication.

The TAR element is a viral regulatory element extending from +1 to +60 in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, which is critical for activation by the transactivator protein Tat. Jurkat cell lines chronically infected with viruses containing HIV-1 TAR element mutations are extremely defective for both gene expression and replication. We previously demonstrated that viruses containing mutations of the TAR RNA stem, bulge, or loop structures have 200- to 5,000-fold-reduced levels of gene expression compared with lymphoid cells harboring wild-type virus. In this study, we characterized several Jurkat cell lines infected with TAR element mutant viruses which spontaneously produced culture supernatants with wild-type-like levels of reverse transcriptase activity. These viral supernatants were used to infect Jurkat cells, and following PCR amplification of the viral long terminal repeats, their DNA sequences were analyzed. This analysis demonstrated that revertant viruses isolated from these cell lines retained the original TAR mutations but also contained additional compensatory mutations within TAR. In gel retardation analysis, recombinant Tat protein bound to higher levels to in vitro-transcribed revertant TAR RNAs than the original TAR RNA mutants. Both the original and revertant TAR elements were inserted into both chloramphenicol acetyltransferase reporter and HIV-1 proviral constructs and assayed following transfection of Jurkat cells. Constructs containing revertant TAR element mutations were capable of strong activation by Tat in contrast to constructs containing the original TAR mutations. Analysis of the secondary structure of TAR RNA sequences suggested that TAR RNA structures which differed from that of wild-type TAR were still capable of strong activation in response to Tat. These results further define critical sequences in TAR RNA that are required for tat activation. In addition, since TAR structures with lower free energy that preserve the loop and bulge structures may be favored over fully formed TAR RNA with higher stable free energy, these results implicate nascent RNA rather than the fully formed TAR RNA structure as the target for tat activation.

Characterization of conditions for the activation of endogenous guinea pig retrovirus in cultured cells by 5-bromo-2'-deoxyuridine.

Human endogenous retroviral sequences recently have been shown to be associated with breast cancer and some leukemias. These retroviral sequences have similarities to an endogenous retrovirus expressed in guinea pigs. The conditions for activation of this guinea pig retrovirus (GPRV) in cultured guinea pig embryo (GPE) cells using 5-bromo-2'-deoxyuridine (BrdU) was investigated. These studies employed the reverse transcriptase activity (RT) assay and electron microscopy (EM), in conjunction with Northern blot analysis that utilized a 2.6 kb GPRV-specific cDNA probe. Contrary to published studies, dexamethasone at concentrations ranging from 10(-8) to 10(-5) M appeared to play a minimal role in enhancing the production of GPRV. Following a 6 hr incubation with BrdU, GPRV mRNA was present in cultured GPE cells. Extracellular virion release was also observed by EM 12 hr later, although RT activity was not detected. All three methods detected viral expression at 48 hr after the addition of the drug. Additionally, after 6 hr exposure to BrdU, detectable RT and mRNA levels were maintained through 44 days after the removal of BrdU in a stationary culture condition and through 31 days in cultures that were subcultured weekly in media not containing BrdU. Low levels of extracellular viruses were detected in these cultures by electron microscopy through 49 days. Therefore, after only a 6 hr exposure to BrdU was extracellular GPRV detected 12 hr after drug removal and virus production continued for up to 49 days. This study provides information about an animal endogenous retroviral system that may be used as a model for the study of human endogenous retroviruses.

Analysis of HIV type 1 reverse transcriptase expression in a human cell line.

The functional analysis of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) subunits on transient and constitutive expression, in the absence or presence of the HIV-1 protease (PR) expression, in a human cell line is described. HIV-1 RT is a heterodimer composed of a 51-kDa subunit (p51) and a 66-kDa subunit (p66). Cloning and expression of the RT region of the HIV-1 pol gene in the HT-1080 human fibrosarcoma cell line yielded p66 without any detectable p51 and a low level of RT activity could be measured. Transient expression of PR and RT in cis generated p51 and p66, but when RT and PR were expressed in trans only p66 was produced. Attempts to establish a stable cell line expressing the PR-RT region of the pol gene were hampered by an apparent intolerance of HT-1080 cells to the HIV-1 PR expression. Therefore, to generate p51 independent of PR expression, the 51-kDa subunit was cloned separately. p51 lacked detectable RT activity. Coexpression of p51 and p66 resulted in a dramatic increase in RT activity. Stable HT-1080 cells producing both p51 and p66 exhibited on average a 15-fold increase in RT activity compared to the parental cell line. Immunofluorescence revealed a diffuse cytoplasmic localization of p51 and p66. To date, this is the first example of a human cell line that is constitutively expressing HIV-1 RT in the absence of HIV-1 infection.

Association of a Retrovirus with a Wasting Condition in the Swedish Moose

A wasting disease in moose (Alces alces) has killed more than one thousand animals in a densely populated, limited geographical area in Sweden. To investigate the cause, local environmental conditions were studied and infectious agents, mainly viruses, were looked for. A virus, identified as a retrovirus, has repeatedly been isolated from diseased animals by cocultivation of their leukocytes with fetal moose kidney cells. The virus has the characteristics of a member of the Oncovirinae subfamily, as deduced from its morphology from EM studies, reverse transcriptase activity, cell transforming properties, and serological cross-reactivity with other oncoviruses. Striking features of the disease are the poor and transient capacity of affected moose to respond serologically in addition to the frequent presence of high levels of reverse transcriptase activity in their sera.

Analysis of long-term viral expression in CEM cells persistently infected with non syncytium-inducing HIV-1 strains.

CEM cells were infected with three HIV-1 non syncytium-inducing (NSI) strains obtained from AIDS patients or seropositive individuals. The surviving cells were followed for several months in the persistently infected cultures designated 65870/CEM, 65871/CEM and 3929/CEM, and analyzed for virus expression using light and electron microscopy, immunofluorescence, reverse transcriptase assay, polymerase chain reaction amplification (PCR), nucleic acid hybridization and flow cytometry. The virus isolates induced relatively few syncytia and other cytopathic effects in the corresponding cell lines and the number of cells positive for virus expression never rose above 44%. Distinct peaks of antigen-positive cells were obtained, coincident with high levels of reverse transcriptase activity. The cultures were strongly resistant to superinfection by laboratory strain Lai, with the exception of 65870/CEM which expressed HIV antigens in up to 15% of the cells for a few days. However, cell lysis was minimal in all cases. After long-term cultivation of the three cultures, no antigen-positive cells were detected and no trace of virus expression could be observed. The remaining cells consisted entirely of CD4-negative cells. PCR analyses indicated that cells harboring a provirus were progressively eliminated from the cultures, leaving only virus-free cells. In this system, cells carrying a latent provirus survive for a limited period of time before virus activation induces cell lysis. These results suggest that at least three types of cells exist in the CEM cell line: CD4-positive cells which are rapidly killed by the virus, a second type harboring a latent viral genome after the infection and which grow normally until activation of the resident genome by external or internal signal(s), and a third type which represents rare CD4-negative cells present in the initial CEM population and which are selected for by the NSI isolates. This is the first study documenting specific interactions between NSI strains of HIV-1 and distinct subpopulations of CEM cells grown as a single cell culture.

In vitro non-productive infection of purified natural killer cells by the BRU isolate of the human immunodeficiency virus type 1.

Highly purified natural killer (NK) cell lines and clones, displaying the typical phenotype, morphology and function and obtained from healthy blood donors, were infected in vitro with the BRU isolate of human immunodeficiency virus type 1 (HIV-1). There was no significant increase in reverse transcriptase activity and levels of p24 antigen in the supernatants, but positive staining was observed using an immunogold technique with polyclonal anti-HIV-1 antibodies. When infected NK cells were co-cultivated with autologous non-infected CD4+ mitogen-activated cells, significant levels of reverse transcriptase activity and p24 antigen in supernatants were detected. Giant syncytial cells and a high number of mature virion particles were also evident. When NK cell lines or clones from HIV-1-infected patients were studied, neither the presence of p24 antigen nor reverse transcriptase activity was detected in the supernatants after stimulation with mitogens, cytokines or co-culture with allogeneic CD4+ mitogen-activated cells. PCR studies did not detect HIV-1 genes in freshly purified NK cells, cell lines or clones from infected patients. Taken together these results suggest that (i) normal NK cells can be infected in vitro by the HIV-1 BRU isolate in a non-productive fashion, (ii) PCR with NK cell DNA of HIV-1-infected patients indicates that in vivo few of these cells, if any, are infected by HIV-1 and (iii) the mechanisms responsible for the impairment of NK cell function during HIV-1 infection remain to be determined and are probably not related to a direct cytopathic effect of the virus.

The nonmyristylated Pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with Pr55gag and is incorporated into viruslike particles.

The expression of the pol gene of human immunodeficiency virus type 1 occurs via a ribosomal frameshift between the gag and pol genes. The resulting protein, a Gag-Pol polyprotein, is produced at a level 5 to 10% of that of the Gag protein. The Gag-Pol polyprotein is incorporated into virions and provides viral protease, reverse transcriptase, and integrase, which are essential for infectivity. It is generally believed that the Gag-Pol polyprotein is incorporated into virions via interaction with the Gag protein, although the details of the mechanism are unknown. To further study this problem, we have constructed a human immunodeficiency virus type 1 proviral genome which overexpresses the Gag-Pol polyprotein (Pr160gag-pol). Transfection of this proviral genome (pGPpr-) into COS-1 cells resulted in the expression of full-length Pr160gag-pol polyprotein. Although the majority of the Pr160gag-pol was confined to the cells, low levels of reverse transcriptase activity were detectable in the cell supernatants. The cotransfection of pGPpr- with a second plasmid which expresses only the Pr55gag precursor (pGAG) resulted in a significantly higher level of Pr160gag-pol in the medium of transfected cells. Sedimentation analysis using sucrose density gradients demonstrated that most Pr160gag-pol was found in fractions corresponding to the density of virion particles, indicating that the Pr160gag-pol polyprotein was released in association with a Pr55gag viruslike particle. To further characterize the requirements for the release, a mutation was constructed to express an unmyristylated Pr160gag-pol polyprotein. Coexpression with Pr55gag demonstrated that the unmyristylated Pr160gag-pol was also incorporated into virion particles. Subcellular fractionation experiments revealed that the distributions of the Pr160gag-polmyr- and Pr160gag-pol in the membrane and cytosol were similar under low- or high-ionic-strength conditions. Taken together, these results suggest that myristylation of the Pr160gag-pol polyprotein is not required for the interaction with the Pr55gag necessary for packaging into a viruslike particle.

Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1 LAI, HIV-1 MAL, and HIV-1 ELI

The construction and preliminary biological characterization of three molecular clones of human immunodeficiency virus type 1 (HIV-1) are reported: HIV-1 LAI from a French man with AIDS, HIV-1 MAL from a Zairian boy with ARC, and HIV-1 ELI from a Zairian woman with AIDS. All three sequences were found to code for infectious viruses. Both the host range and the kinetics of infection in CD4 + cells were different for the three viruses. Virus derived from each molecular clone was infectious on peripheral blood mononuclear cells (PBMC), although LAI and ELI displayed more rapid growth kinetics than MAL. The viruses had different tropisms and growth kinetics in six cell lines. LAI was infectious in all of the cell lines and produced high levels of reverse transcriptase activity. MAL and ELI had more restricted tropisms: MAL could only replicate on SupT1, whereas ELI grew on Jurkat and MT-4, was delayed on CEM and H9, and was unable to infect U937 cells. In addition, we observed that both the replicative capacity and the cell tropism of viruses could change after passage through some established cell lines. These results suggest that the genotypes of some viruses in vitro are not stable and that selection for growth can cause the fairly rapid appearance of variants with increased growth potential.

Replication of an acutely lethal simian immunodeficiency virus activates and induces proliferation of lymphocytes

A variant of simian immunodeficiency virus from sooty mangabey monkeys (SIVsmm), termed SIVsmmPBj14, was previously identified and shown to induce acute disease and death within 1 to 2 weeks of inoculation of pig-tailed macaques and mangabey monkeys (P. N. Fultz, H. M. McClure, D. C. Anderson, and W. M. Switzer, AIDS Res. Hum. Retroviruses 5:397-409, 1989). SIVsmmPBj14 differed from its parent virus, SIVsmm9, not only in pathogenicity but also in multiple in vitro properties. As a first approach to understanding the biological and molecular mechanisms responsible for the acute disease and death induced by this variant, virus-host cell interactions of SIVsmmPBj14 and SIVsmm9 were studied. Initial rates of replication of the two viruses were identical in primary peripheral blood mononuclear cells (PBMC) from normal pig-tailed macaques and mangabey monkeys, but SIVsmmPBj14 infection always resulted in higher yields of virus than did SIVsmm9 infection, as assessed by levels of reverse transcriptase activity in culture supernatants. Surprisingly, despite its cytopathicity for macaque and mangabey CD4+ cells, replication of SIVsmmPBj14 was accompanied by up to 10-fold increases in number of viable cells compared with cell numbers in uninfected or SIVsmm9-infected cultures. Furthermore, SIVsmmPBj14 was shown to infect and replicate in resting PBMC just as efficiently as in mitogen-stimulated PBMC, irrespective of whether exogenous interleukin-2 (IL-2) or antibodies that neutralized IL-2 were added to culture media. Accumulation of virus in culture supernatants of resting PBMC preceded by several days the appearance of activated cells which expressed the IL-2 receptor alpha subunit (CD25), suggesting that activation of cells was not essential for replication. The ability to activate and to induce simian PBMC to proliferate appeared specific for the acutely lethal variant because incorporation of [3H]thymidine by PBMC from naive animals was observed only upon incubation with concentrated, heat-inactivated SIVsmmPBj14 and not with other viruses. Both CD4(+)- and CD8(+)-enriched cell populations proliferated in response to SIVsmmPBj14. These results are consistent with in vivo observations and suggest that the abilities both to replicate in resting cells and to induce lymphocytes to proliferate may contribute to the extreme virulence of SIVsmmPBj14.