Recurrent emergence of structural variants of LTR retrotransposon CsRn1 evolving novel expression strategy and their selective expansion in a carcinogenic liver fluke, Clonorchis sinensis.

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy-like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag-pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini, the closest neighbor of C. sinensis, whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome.

HBV polymerase overexpression due to large core gene deletion enhances hepatoma cell growth by binding inhibition of microRNA-100.

Different types of hepatitis B virus (HBV) core gene deletion mutants were identified in chronic hepatitis B patients. However, their clinical roles in different stages of natural chronic HBV infection remained unclear. To address this issue, HBV core genes were sequenced in three gender- and age-matched patient groups diagnosed as chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC), respectively. Functional analysis of the identified mutants was performed. A novel type of large-fragment core gene deletion (LFCD) was identified exclusively in HCC patients and significantly associated with unfavorable postoperative survival. The presence of LFCDs resulted in generation of precore-polymerase fusion protein or brought the polymerase reading frame under direct control of HBV precore/core promoter, leading to its over-expression. Enhanced cell proliferation and increased tumorigenicity in nude mice were found in hepatoma cells expressing LFCDs. Because of the epsilon-binding ability of HBV polymerase, we hypothesized that the over-expressed polymerase carrying aberrant amino-terminal sequence could bind to cellular microRNAs. Screening of a panel of microRNAs revealed physical association of a precore-polymerase fusion protein with microRNA-100. A binding inhibition effect on microRNA-100 by the precore-polymerase fusion protein with up-regulation of its target, polo-like kinase 1 (PLK1), was discovered. The binding inhibition and growth promoting effects could be reversed by overexpressing microRNA-100. Together, HCC patients carrying hepatitis B large-fragment core gene deletion mutants had an unfavorable postoperative prognosis. The growth promoting effect was partly due to polymerase overexpression, leading to binding inhibition of microRNA-100 and up-regulation of PLK1.

Large-scale production and structural and biophysical characterizations of the human hepatitis B virus polymerase.

UNLABELLED: Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies--broad-spectrum antivirals--are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of ß-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCE: The viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.

Modulation of ribosomal frameshifting frequency and its effect on the replication of Rous sarcoma virus.

Programmed -1 ribosomal frameshifting is widely used in the expression of RNA virus replicases and represents a potential target for antiviral intervention. There is interest in determining the extent to which frameshifting efficiency can be modulated before virus replication is compromised, and we have addressed this question using the alpharetrovirus Rous sarcoma virus (RSV) as a model system. In RSV, frameshifting is essential in the production of the Gag-Pol polyprotein from the overlapping gag and pol coding sequences. The frameshift signal is composed of two elements, a heptanucleotide slippery sequence and, just downstream, a stimulatory RNA structure that has been proposed to be an RNA pseudoknot. Point mutations were introduced into the frameshift signal of an infectious RSV clone, and virus replication was monitored following transfection and subsequent infection of susceptible cells. The introduced mutations were designed to generate a range of frameshifting efficiencies, yet with minimal impact on encoded amino acids. Our results reveal that point mutations leading to a 3-fold decrease in frameshifting efficiency noticeably reduce virus replication and that further reduction is severely inhibitory. In contrast, a 3-fold stimulation of frameshifting is well tolerated. These observations suggest that small-molecule inhibitors of frameshifting are likely to have potential as agents for antiviral intervention. During the course of this work, we were able to confirm, for the first time in vivo, that the RSV stimulatory RNA is indeed an RNA pseudoknot but that the pseudoknot per se is not absolutely required for virus viability.

Expression and purification of a functional human hepatitis B virus polymerase.

AIM: To identify a method for efficient large-scale purification of functional hepatitis B virus polymerase (HBV-Pol) without addition of cellular factors. METHODS: Full-length HBV-Pol (843 amino acids) tagged with 5' end Polyhistidine was expressed at a high level in an Escherichia coli (E. coli) system. Sodium dodecyl sulfate lysis buffer was utilized to dissolve insoluble HBV-Pol, and Ni-NTA resin affinity chromatography was utilized for HBV-Pol purification. Most recombinant HBV-Pol was eluted with 100 mmol/L imidazole in the presence of NP-40, a weak detergent that keeps HBV-Pol in solution. A reducing agent was utilized throughout the purification steps to keep soluble HBV-Pol from redundant disulfide bond formation. RESULTS: The large-scale production of functional intact human HBV-Pol was achieved in an E. coli expression system. Purified HBV-Pol showed stable reverse transcriptase activity and DNA polymerase activity. The purified protein was of high purity and had stable reverse transcriptase activity. CONCLUSION: Large-scale production of HBV-Pol in pure form should facilitate crystallization and detailed analysis of the structure and mechanism of HBV-Pol. Ability of this purification approach to obtain human HBV-Pol in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.

Modulation of hepatitis B virus replication by expression of polymerase-surface fusion protein through splicing: implications for viral persistence.

In hepatitis B virus (HBV)-infected livers and -transfected hepatoma cells, spliced transcripts are not essential for HBV replication. However, their ability to modulate HBV replication has not been clearly elucidated. In the current study, we found that the polymerase-surface (PS) fusion protein, generated from a spliced HBV transcript, colocalized with the nuclear pore complex, vimentin, microtubules, and the endoplasmic reticulum (ER) in the perinuclear region of transfected cells. We found that PLC/PRF/5-hepatoma cells expressed PS transcript and PS protein. Hepatitis B surface antigen (HBs) secretion, core particle formation, and HBV DNA synthesis were inhibited by the expression of PS transcript and PS protein, and by expression of PS transcript alone, suggesting that HBV replication is modulated by splicing. Our results suggest that splicing may be one of the outcomes of the host-virus interaction.

Targeted screening of SiRNA directed HBV polymerase gene for effective inhibition of HBV expression.

In order to screen potential mRNA locations of P gene in which targeting siRNAs can effectively inhibit HBV expression, 5 recombinant plasmids containing 4 targeting-specific siRNA fragments and a control were prepared and transfected into 2.2.15 cells respectively. The expression levels of HBx mRNA, HBs mRNA and HBc mRNA were detected by RT-PCR. The concentrations of the hepatitis B virus antigens, including HBsAg and HBeAg harvested from the culture supernatant of transfected 2.2.15 cells, were measured by ELISA. X protein was tested by Western blot. The results showed that four siRNAs against distinct mRNA locations of HBV polymerase gene had different inhibitory effects on their targeted mRNA. The plasmid-derived psiRNA1 and psiRNA2 could effectively inhibit the transcription and translation of HBs gene, whereas the inhibitory efficiency of psiRNA3, psiRNA4 for HBe gene was much higher than that of psiRNA1 and psiRNA2. In comparison to the rest of psiRNAs in this study, psiRNA4 was the most effective to suppress the transcription and translation of HBx. It is suggested that siRNA can be considered as a powerful therapeutic agent for reducing HBV expression. The siRNAs against HBV polymerase are effective largely depending on the location of targeted sites. To enhance inhibitory efficiency, hunting for high effective target in polymerase gene is necessary and feasible.

[Intergration and epression of porcine endogenous retrovinus in the immortal cell line of Banna Minipig Inberd Line-Mesenhymal Stem Cells].

OBJECTIVE: To detect the integration and expression of porcine endogenous retrovirus (PERV) in the immortal cell line of Banna Minipig Inbred Line-Mesenchymal Stem Cells (BMI-MSCs). METHODS: DNA and total RNA of the immortal cell line of BMI-MSCs were extracted and PCR, RT-PCR were performed to detect PERV-gag, pol and env gene, and the type of PERV was also detected. RESULTS: PERV-gag, pol and env gene were all detected in the primary culture and immortal cell line (passage 150 and passage 180) of BMI-MSCs, and the type of PERV was PERV-A, B. Functional expression of PERV-gag and pol mRNA was also detected. CONCLUSION: In this laboratory, PERV was not lost during the proceeding of pig inbred and since has been in long-term culture of pig cells in vitro. PERV has integrated into the genome of its natural host, and virus mRNA can effectively express. So it is very essential to evaluate the possibility of xenozoonoses in pig-to-human xenotransplantation.

Involvement of PKR and RNase L in translational control and induction of apoptosis after Hepatitis C polyprotein expression from a vaccinia virus recombinant.

BACKGROUND: Hepatitis C virus (HCV) infection is of growing concern in public health with around 350 million chronically infected individuals worldwide. Although the IFN-alpha/rivabirin is the only approved therapy with 10-30% clinical efficacy, the protective molecular mechanism involved during the treatment is still unknown. To analyze the effect of HCV polyprotein expression on the antiviral response of the host, we developed a novel vaccinia virus (VV)-based delivery system (VT7-HCV7.9) where structural and nonstructural (except part of NS5B) proteins of HCV ORF from genotype 1b are efficiently expressed and produced, and timely regulated in mammalian cell lines. RESULTS: Regulated transcript production and viral polypeptide processing was demonstrated in various cell lines infected with the recombinant VT7-HCV7.9, indicating that the cellular and viral proteolytic machineries are functional within these cells. The inducible expression of the HCV polyprotein by VV inhibits the synthesis of both host and viral proteins over the time and also induces apoptosis in HeLa and HepG2-infected cells. These effects occur accompanying with the phosphorylation of the translation initiation factor eIF-2alpha. In cells co-infected with VT7-HCV7.9 and a recombinant VV expressing the dominant negative eIF-2alpha-S51A mutant in the presence of the inductor isopropyl-thiogalactoside (IPTG), protein synthesis is rescued. The IFN-inducible protein kinase PKR is responsible for the translational block, as demonstrated with PKR-/- and PKR +/+ cell lines. However, apoptosis induced by VT7-HCV7.9 is mediated by the RNase L pathway, in a PKR-independent manner. CONCLUSION: These findings demonstrate the antiviral relevance of the proteins induced by interferon, PKR and RNase L during expression from a VV recombinant of the HCV polyprotein in human cell lines. HCV polyprotein expression caused a severe cytopathological effect in human cells as a result of inhibition of protein synthesis and apoptosis induction, triggered by the activation of the IFN-induced enzymes PKR and RNase L systems. Thus, the virus-cell system described here highlights the relevance of the IFN system as a protective mechanism against HCV infection.

DNA vaccines prime CD8+ T cell responses to epitopes of viral antigens produced from overlapping reading frames of a single coding sequence.

A hepatitis B virus (HBV)-derived sequence that encodes the 832-residue polymerase (Pol) protein of HBV in the primary open reading frame (ORF), and the three (large, middle and small) hepatitis B surface antigen (HBsAg) variants in an alternative ORF was used. This sequence was cloned into expression vectors in which Pol was expressed under heterologous (HCMV, SV40 or metallothionin) promoter control. Some Pol-encoding vectors coexpressed Pol as well as readily detectable amounts of HBsAg. Efficient HBsAg expression depended on endogenous HBV promoter sequences but was apparently also facilitated by heterologous promoter sequences located upstream of the HBV Pol sequence. DNA immunization of mice efficiently coprimed CD8(+) T cell responses to epitopes of Pol and HBsAg. Over expression of Pol (using an hsp73-facilitated expression system) did not correlate with the immunogenicity of the K(d)/Pol(140-148) epitope. Immunodominant L(d)-restricted CD8(+) T cell responses to HBsAg down-modulated priming of CD8(+) T cell responses to other HBsAg epitopes but not to the K(d)/Pol(140-148) epitope. Different antigens transcribed from alternative reading frames of a single sequence in a DNA vaccine can thus efficiently prime multispecific T cell responses.

Functionally impaired HIV-specific CD8 T cells show high affinity TCR-ligand interactions.

We eventually isolated two different clonotypic CD8 T cell subsets recognizing an HIV Pol-derived epitope peptide (IPLTEEAEL) in association with HLA-B35 from a chronic HIV-infected patient. By kinetic analysis experiments, the subsets showed a >3-fold difference in half-lives for the HLA tetramer in complex with the Pol peptide. In functional assays in vitro and ex vivo, both subsets showed substantial functional avidity toward peptide-loaded cells. However, the high affinity subset did not show cytolytic activity, cytokine production, or proliferation activity toward HIV-infected cells, whereas the moderate affinity one showed potent activities. Furthermore, using ectopic expression of each of the TCR genes into primary human CD8 T cells, the CD8 T cells transduced with the high affinity TCR showed greater binding activity toward the tetramer and impaired cytotoxic activity toward HIV-infected cells, corroborating the results obtained with parental CD8 T cells. Taken together, these data indicate that impaired responsiveness of T cells toward HIV-infected cells can occur at the level of TCR-ligand interactions, providing us further insight into the immune evasion mechanisms by HIV.

Role of the C terminus of foamy virus Gag in RNA packaging and Pol expression.

Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol proteins are expressed independently of one another, and both proteins undergo single cleavage events. Thus, the mature FV Gag protein does not consist of the matrix, capsid, and nucleocapsid (NC) proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol protein.

Avipox-based simian immunodeficiency virus (SIV) vaccines elicit a high frequency of SIV-specific CD4+ and CD8+ T-cell responses in vaccinia-experienced SIVmac251-infected macaques.

The ability of ALVAC- or fowlpox-based simian immunodeficiency virus (SIV) vaccines to boost SIV-specific CD4+ and CD8+ T-cell responses was tested in 10 vaccinia-experienced macaques infected with SIVmac251. The CD8+ T-cell response to the dominant Gag(181-189) CM9 was quantitated in seven Mamu-A*01-positive macaques by tetramer staining, by ex vivo cytotoxic T-lymphocyte (CTL) activity, and by intracellular cytokine staining (ICS) with the specific Gag(181-189) CM9 peptide. The overall CD8+ T-cell response to Gag was assessed using a peptide pool encompassing the entire Gag protein followed by measurement of TNF-alpha production in ICS assay. Similarly, virus-specific CD4+ T-cell responses were measured by ICS for TNF-alpha following stimulation with the Gag-overlapping peptide and by proliferative response following stimulation with purified p27 Gag. The two vaccine modalities effectively boosted both CD4+ and CD8+ SIV-specific T-cell response despite prior exposure to the vaccinia-derivative NYVAC vector, suggesting that sequential boosting with either avipox-based vector vaccine candidate is a realistic approach in immune therapy of human immunodeficiency virus type 1 (HIV-1)-infected individuals.

An endogenous retrovirus derived from human melanoma cells.

We show that human melanoma cells produce retrovirus-like particles that exhibit reverse transcriptase activity, package sequences homologous to human endogenous retrovirus K (HERV-K), and contain mature forms of the Gag and Env proteins. We also demonstrate expression of the pol gene and of Gag, Env, and Rec proteins in human melanomas and metastases but not in melanocytes or normal lymph nodes. The data suggest that expression of retroviral genes and production of retroviral particles is activated during development of melanoma.

Proteolytic processing of foamy virus Gag and Pol proteins.

The foamy viral proteases (FV PRs) are set apart from other retroviral processing enzymes by unique features. The first remarkable property is that FV PRs are enzymatically active as high-molecular-mass Pro-Pol proteins. Hence there exist multiple forms of active FV PRs that likely contribute to cleavage site specificity. A FV PR of low molecular size is not detectable in purified virions, in contrast to PRs of other retroviruses that are found in virus particles. Because the major part of Pol remains attached to the amino-terminal enzymatically active PR protein region, the FV-specific way of expressing Pro-Pol polyproteins from a pol-specific transcript provides for the incorporation of Pro-Pol and IN into virus particles. Proteolytic processing of Gag and Pol proteins is incomplete and delayed. Another novel feature is that the catalytic center of the active dimers of cat FV PR consists of D-S/T-Q instead of D-S/T-G, an unprecedented feature of this enzyme. The temporal and spatial control and the factors that regulate FV PRs remain to be elucidated.

Particle assembly and genome packaging.

Foamy virus (FV) replication is distinct from that of all other retroviruses in many respects, including viral assembly. In fact, the viral assembly pathway is rather similar to that of hepadnaviruses such as hepatitis B virus. Foamy virus Gag does not contain landmark retroviral assembly domains such as the major homology region, Cys-His boxes, or a defined M domain. Like hepadnaviruses, the FV Gag protein is not cleaved and contains arginine-rich regions at the carboxyl terminus. In addition, egress of FV particles requires presence of the envelope glycoproteins. Finally, the cis-acting sequences in the FV genome required for genome incorporation, although poorly defined, differ in location from other retroviruses.

Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli.

We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli. We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins. The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors. The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation. However, nondenaturing and partially denaturing activity gel analyses showed two active bands of approximately 260 kDa and approximately 130 kDa, respectively. Furthermore, in the presence of the encapsidation signal RNA template (HBV epsilon RNA), the approximately 260-kDa active band was gradually converted to approximately 130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV epsilon RNA. These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.

Hematologic recovery in mice transplanted with bone marrow stem cells expressing anti-human immunodeficiency virus genes.

We have used a mouse bone marrow transplantation (BMT) model to study the safety of retrovirus-mediated transfer of anti-HIV genes (RevM10 and HIV-1 pol antisense) into hematopoietic stem/progenitor cells (HSPCs). In particular, we have monitored the hematologic recovery post-BMT and transgene expression in myeloid and lymphoid lineages, and analyzed tissue sections for evidence of any transgene-related pathological condition. Expression of anti-HIV genes had no effect on kinetics of hematologic recovery post-BMT. The average time to reach 20% of normal cell counts was 15-17 days for white blood cells and 12-14 days for platelets, and the average time to reach complete recovery was 42-56 days for leukocytes and 104-161 days for platelets. Hematocrit levels were not significantly affected by irradiation and transplantation procedures. Donor chimerism was uniformly > or =90% in all transplanted animals. At 4-5 weeks post-BMT transgene expression was detected in peripheral blood leukocytes in 100% of the animals and ranged from 4.5 to 44.7%. In a majority of the animals the percentage of transgene-expressing cells in circulation decreased over time but remained detectable for the length of the study (>6 months). Expression was detected in all analyzed cell lineages (RBCs, platelets, monocytes, granulocytes, and T and B cells). Relative counts of various leukocytes (Mac1+ monocytes, Gr1+ granulocytes, CD3+ T cells, and B220+ B cells) were normal. There were no treatment-related histopathological changes in a wide range of tissues examined. In addition, there were no treatment effects on differential leukocyte counts, and morphology of peripheral blood and bone marrow brush smears. In summary, transfer and expression of the RevM10 and the HIV-1 antisense genes into hematopoietic stem/progenitor cells in vivo appears safe. We propose that the mouse bone marrow transplantation model could be used to evaluate some safety aspects of HSPC-based gene therapies.

Regulation of bovine leukemia virus tax and pol mRNA levels by interleukin-2 and -10.

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.

Characterization of gag and pol sequences from long-term survivors of human immunodeficiency virus type 1 infection.

We previously identified a group of 10 long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection. Extensive biological analysis revealed that some of these individuals do well, at least in part, because they possess weakened or attenuated viruses. Also, previously, to determine the genotype associated with the attenuated phenotype in vivo, we characterized nef, vif, vpr, vpu, env, and LTR in our cohort of LTS. In this study, we analyzed gag and pol genes derived from eight individuals in our cohort. For each subject multiple full-length gag and pol clones were obtained for analysis. In most cases, the sequences derived from the LTS had an intact open reading frame. At the protein level, there were no discernible differences between the sequences derived from LTS and those derived from patients with AIDS. Thus, no common defect in gag and pol was found in our cohort. One individual (subject SF), however, had only Gag-defective proviral sequences (10 of 10) in his peripheral blood mononuclear cells. Furthermore, longitudinal studies of the samples collected from SF over a 2-year period showed that all p17 gag clones sequenced (24 of 24) were defective due to G-to-A hypermutations. This viral defect in Gag may provide the molecular basis for this individual's extremely low viral load and long-term asymptomatic state. These results, together with previous findings in our LTS cohort, reinforce the notion that it is unlikely that a single common viral genetic determinant accounts for the lack of disease progression in all cases. Multiple host and viral factors undoubtedly contribute to the well-being of LTS of HIV-1 infection.