Molecular cloning of Kuruma shrimp Marsupenaeus japonicus endonuclease-reverse transcriptase and its positive role in white spot syndrome virus and Vibrio alginolyticus infection.

This study investigated the function of endonuclease-reverse transcriptase (mjERT) in Marsupenaeus japonicus. The 1129 bp cDNA sequence of mjERT was cloned from M. japonicus using rapid amplification of cDNA ends (RACE) PCR, and RT-qPCR analysis indicated that mjERT was highly expressed in the gills and hepatopancreas of M. japonicus. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could enhance the expression of mjERT. When mjERT was inhibited, immune genes such as toll, p53, hemocyanin and tumor necrosis factor-α (TNF-α) were significantly down-regulated (P < .01) in the hemocytes of shrimp, while myosin was significantly up-regulated (P < .01). We demonstrated that mjERT is very important for the progression of WSSV infection and that the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimps was significantly increased following mjERT RNA interfere (RNAi). Apoptosis data provided information to suggest that mjERT-dsRNA challenge caused less apoptosis in hemocytes in both the disease-free and viral group. We also revealed that mjERT-dsRNA treatment resulted in a lower phagocytosis rate in the hemocytes of V. alginolyticus-challenged shrimp. Finally, we found that the absence of mjERT had an significantly negative impact upon shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating a regulative role for mjERT in the innate immunity of shrimp in response to pathogenic infection. In summary, we concluded that mjERT might promote the anti-WSSV immune response of shrimp by regulating apoptosis, PO activity, THC and SOD activity, and also exert a positive role in the immune response against V. alginolyticus by regulating phagocytosis, SOD activity, PO activity and THC.

High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo.

The low fidelity of HIV replication facilitates immune and drug escape. Some reverse transcriptase (RT) inhibitor drug-resistance mutations increase RT fidelity in biochemical assays but their effect during viral replication is unclear. We investigated the effect of RT mutations K65R, Q151N and V148I on SIV replication and fidelity in vitro, along with SIV replication in pigtailed macaques. SIVmac239-K65R and SIVmac239-V148I viruses had reduced replication capacity compared to wild-type SIVmac239. Direct virus competition assays demonstrated a rank order of wild-type>K65R>V148I mutants in terms of viral fitness. In single round in vitro-replication assays, SIVmac239-K65R demonstrated significantly higher fidelity than wild-type, and rapidly reverted to wild-type following infection of macaques. In contrast, SIVmac239-Q151N was replication incompetent in vitro and in pigtailed macaques. Thus, we showed that RT mutants, and specifically the common K65R drug-resistance mutation, had impaired replication capacity and higher fidelity. These results have implications for the pathogenesis of drug-resistant HIV.

Enhanced expression of LINE-1-encoded ORF2 protein in early stages of colon and prostate transformation.

LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types, while being silenced in most differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promotes differentiation in neoplastic cells, indicating that high endogenous RT activity promotes cancer growth. Here we investigate the expression of L1-ORF2p in several human types of cancer.We have developed a highly specific monoclonal antibody (mAb chA1-L1) to study ORF2p expression and localization in human cancer cells and tissues.We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate tissues shows ORF2p expression in preneoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively.Our results show that L1-ORF2p is overexpressed in tumor and in preneoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early diagnostic biomarker.

Priming with recombinant auxotrophic BCG expressing HIV-1 Gag, RT and Gp120 and boosting with recombinant MVA induces a robust T cell response in mice.

In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCGΔpanCD) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCGΔpanCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8(+) T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 10(5) cfu to 10(7) cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCGΔpanCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-γ-spot forming cells than mice primed with rBCGΔpanCD containing an empty vector control. Priming with the individual rBCGΔpanCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4(+) and CD8(+) T cells producing IFN-γ and TNF-α and CD4(+) cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix.

Evolutionarily conserved epitopes on human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus reverse transcriptases detected by HIV-1-infected subjects.

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV(+)) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV(+) subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8(+) T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8(+) T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV(+) subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine.

Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1% variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9% nucleotide conservation in the overlapping region vs 41.2% in the non-overlapping region; P<0.001), with a consequently lower rate of synonymous substitution within the overlapping region [median(interquartile range)dS=3.1(1.5-7.4) vs 20.1(10.6-30.0); P=3.249×10(-22)]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6%), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8% in HIV-HBV co-infection vs 21.5% in mono-infected patients; P=0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.

Immunogenicity of a recombinant measles-HIV-1 clade B candidate vaccine.

Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 10(5) TCID(50) of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.

Autoimmunogenicity of the helix-loop-helix DNA-binding domain.

Nonimmunogenic character of native DNA, and its high immunogenicity when presented in complex with the DNA-binding proteins indicate that the latter might contain molecular triggers of anti-DNA response. To find if this is the case, we have evaluated the autoimmunogenic potential of the main DNA-binding domain of HIV-1 reverse transcriptase that belongs to the canonical helix-loop-helix type. BALB/c mice were immunized with a peptide representing the domain, alone or in complex with the fragmented human DNA in the presence of an adjuvant. Mice were assessed for specific antibodies, autoantibodies against a panel of self-antigens; glomerular immunoglobulin deposition; and for the signs of autoimmune disease, such as proteinuria, and changes in the blood components. Immunization with the adjuvanted peptide-DNA complex induced autoantibodies against double-stranded DNA, histones, heterochromatin, and kidney proteins; glomerular IgG and IgA deposition; proteinuria; thrombocytopenia, and anemia. Altogether, this identifies the helix-loop-helix DNA-binding domain as one of the molecular triggers of autoimmunity to DNA and DNA-associated proteins. The experiments cast new light on the role of the DNA-binding retroviral proteins in the induction of autoimmunity, and on the origins of autoimmune complications in the microbial infections in general. It also implies that choosing the DNA-binding proteins as vaccine candidates should be done with precaution.

[Human monoclonal antibody inhibiting reverse transcriptase activity of hepatitis B virus polymerase protein].

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1 x 10(10), human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1 micro g of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15 x 10(-8) M to 1.75 x 10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

Involvement of Hsp90 in assembly and nuclear import of influenza virus RNA polymerase subunits.

Transcription and replication of the influenza virus RNA genome occur in the nuclei of infected cells through the viral RNA-dependent RNA polymerase consisting of PB1, PB2, and PA. We previously identified a host factor designated RAF-1 (RNA polymerase activating factor 1) that stimulates viral RNA synthesis. RAF-1 is found to be identical to Hsp90. Here, we examined the intracellular localization of Hsp90 and viral RNA polymerase subunits and their molecular interaction. Hsp90 was found to interact with PB2 and PB1, and it was relocalized to the nucleus upon viral infection. We found that the nuclear transport of Hsp90 occurs in cells expressing PB2 alone. The nuclear transport of Hsp90 was in parallel with that of the viral RNA polymerase binary complexes, either PB1 and PB2 or PB1 and PA, as well as with that of PB2 alone. Hsp90 also interacted with the binary RNA polymerase complex PB1-PB2, and it was dissociated from the PB1-PB2 complex upon its association with PA. Furthermore, Hsp90 could form a stable PB1-PB2-Hsp90 complex prior to the formation of a ternary polymerase complex by the assembly of PA in the infected cells. These results suggest that Hsp90 is involved in the assembly and nuclear transport of viral RNA polymerase subunits, possibly as a molecular chaperone for the polymerase subunits prior to the formation of a mature ternary polymerase complex.

Development of single-domain recombinant antibodies to reverse transcriptase domain of human hTERT.

This paper describes the development of single-domain recombinant antibodies against human telomerase core protein. A His-tagged hTERT spanning main reverse-transcriptase domain of hTERT was purified from host E. coli and used to immunize BALB/c mice. The VHs (heavy chain variable region genes) were amplified by PCR from total RNA of splenocytes and further induced random mutagenesis by DNA shuffling to enrich the repertoire of VH library. All VHs were cloned into phagemid vectors and displayed to generate 4 x 10(10) phage libraries. The candidates carrying VH domains against hTERT were primarily screened through three times of panning procedure on His-tagged hTERT coated microplates, and specific antibodies were further selected by West-Western blot. Two clones, designated as a3 and b8, were confirmed to interact with the target in the solid-phase assay. DNA sequencing proved their mouse VH origin. The purified single-domain antibody of b8 could not only recognize native hTERT, but also neutralize human telomerase activity on inhibitory assay and b8 showed the stronger suppressive efficacy compared with a3. The data demonstrated that the developed single-domain recombinant antibodies were hTERT-specific with high potential of binding and activity inhibition.

Peptide mapping of feline immunodeficiency virus by IFN-gamma ELISPOT.

Interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) has become an important tool in studying antigen-specific T lymphocyte responses. Soluble peptides can be used to map T-cell epitopes, providing information that is useful in the design and evaluation of vaccines as well as studies of immunopathogenesis. To date, this assay has not been widely utilized in feline immunodeficiency virus (FIV) research. We have developed a feline IFN-gamma ELISPOT assay and used it to determine FIV-specific T-cell epitopes recognized by infected cats. A panel of 331 peptides, 15 amino acids in length and overlapping by 10 residues was synthesized. The peptide library spanned the FIV structural (Gag), envelope (Env), reverse transcriptase (RT), and open-reading-frame A (OrfA) proteins. Initially, 34 pools, containing 7-10 peptides each were screened by IFN-gamma ELISPOT against peripheral blood mononuclear cells (PBMC) from eight cats chronically infected with the NCSU(1) molecular clone of FIV and four uninfected control cats. Individual peptides from pools recognized by FIV+ cats were then evaluated and optimal peptides were combined into pools representing Gag, Env, RT, and OrfA. A higher percentage of FIV infected cats were identified as responders against the peptide pools when using fresh PBMC as compared to cryopreserved PBMC. In vitro restimulation of cryopreserved PBMC with the peptide pools improved the sensitivity of the assay to similar levels as observed from fresh samples. Individual peptides used in the pools were generally found to stimulate CD8+ T-cells more efficiently than CD4+ T-cells. Comparison of the peptide sequences to representative FIV sequences from clades A-D showed conservation was high among Gag and RT peptides, variable among Env peptides and low for OrfA peptides. The IFN-gamma ELISPOT assay and FIV-specific peptide pools we describe here will be useful in assessing cell-mediated responses to experimental FIV vaccines.

HIV infection of primary human T cells is determined by tunable thresholds of T cell activation.

HIV infection of primary human T cells requires T cell activation signals. However, how strength, duration, and quality of TCR signals affect susceptibility of resting human T cells to HIV infection remains poorly understood. We found that the same threshold and duration of antigen signals that lead to optimal T cell activation are required for HIV to progress beyond the level of reverse transcription within resting T cells. Remarkably, sustained cytokine signaling from the IL-2 receptor following TCR triggering was critical in establishing productive infection. While blockade of TCR signaling pathways with inhibitors of the phosphatidylinositol 3-kinase pathway caused a partial pre-integration block, another inhibitor, rapamycin, completely suppressed the infection. In contrast, cyclosporin A or FK506, inhibitors of NFAT, failed to block infection if the T cells were pre-activated. Collectively, these results bring to light significant parallels between successful HIV infection and optimal thresholds of T cell activation. Furthermore, our results underscore the critical role of IL-2 signaling in establishing productive HIV infection. These findings have important implications for our understanding of the complex interplay of HIV with host factors induced upon T cell activation.

The immune control and cell-to-cell spread of human T-lymphotropic virus type 1.

Human T-lymphotropic virus type 1 (HTLV-1) varies little in sequence compared with human immunodeficiency virus type 1 (HIV) and it is difficult to detect HTLV-1 mRNA, proteins or virions in fresh blood. But the strong and chronically activated T cell response to the virus indicates that HTLV-1 proteins are expressed persistently. It now appears that the efficiency of an individual's cytotoxic T cell (CTL) response to HTLV-1 is the chief single determinant of that person's provirus load, which can differ between HTLV-1-infected people by more than 10 000-fold. Progress is now being made towards defining this CTL 'efficiency' in terms of host genetics, T cell function, T cell gene expression and mathematical dynamics. Lymphocytes that are naturally infected with HTLV-1 do not produce enveloped extracellular virions in short-term culture and this has reinforced the erroneous conclusion that the virus is latent. But recent evidence shows that HTLV-1 can spread directly between lymphocytes across a specialized, virus-induced cell-cell contact - a 'viral synapse'. Instead of making extracellular virions, HTLV-1 uses the mobility of the host cell to spread within and between hosts. In this review the evidence is summarized on the persistent gene expression of HTLV-1 in vivo, the role of the immune system in protection and pathogenesis in HTLV-1 infection, and the mechanism of cell-to-cell spread of HTLV-1.

Frequência das mutações que conferem resistência aos anti-retrovirais em um grupo de indivíduos infectados pelo HIV-1, em Salvador, Bahia / Frequency of mutations that confer resistance to antiretroviral drugs in a group of individuals infected by HIV-1 in Salvador, Bahia

A grande variabilidade genética do HIV é um importante obstáculo para a manutenção de terapêuticas de longa eficácia. Assim, o conhecimento sistemático das mutações que conferem resistência aos anti-retrovirais é de grande importância clínica e terapêutica. A partir de uma coleção de plasma de pacientes do Estado da Bahia, realizamos um estudo com o intuito de investigar a freqüência de mutações que conferem resistência aos anti-retrovirais, empregando o ensaio LIPA HIV-1 RT para analisar as mutações no gene da transcriptase reversa e a técnica de RFLP, utilizando a enzima de restrição Hinc lI, para identificar as mutações nas posições 82 e 90 no gene da protease. As amostras selecionadas foram provenientes de pacientes virgens de tratamento (VT; n=22) e de pacientes que estavam sob terapia antiretroviral, na forma de terapia dupla com INTR (TD; n=48) e terapia tripla com INTR e inibidores da protease (TT; n=41). Neste último grupo, realizamos também o teste fenotípico HIV-1 PR/RT Antivirogram TM VIRCO em 17 pacientes. No grupo de pacientes VT, apenas 1/20 (5%) apresentou a mutação M184V no gene da TR, enquanto que 3/22 (13,6%) apresentaram a mutação L90M no gene da protease. Já no grupo TD, encontramos mutações que conferem resistência ao AZT como T215Y/F, K70R e L41M em, respectivamente, 60%, 48% e 48% das amostras analisadas. Mutações que conferem resistência ao 3TC (M184V-44%) e ao ddI (L746%). Nos indivíduos sob TT, as freqüências de mutações no gene da protease foram de 34%-V82A, 15%-L90M e 39%- V82AJL90M. A análise fenotípica mostrou que as drogas menos ativas dos INTR foram AZT e 3TC e dos IP foram saquinavir, ritonavir, indinavir e nelfinavir. Estes resultados estão de acordo com a elevada freqüência de mutações encontradas no gene da protease V82A-60%, L90M-24% e V82AJL90M-65%. Nossos resultados mostram que as mutações genotípicas encontradas têm uma forte correlação com o tipo de drogas anti-retrovirais em uso pelos pacientes incluídos no estudo.

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.

Primate foamy virus Pol proteins are imported into the nucleus.

Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127(Pol), p85(RT/RN) and p40(IN) served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains.

The structural basis for the increased immunogenicity of two HIV-reverse transcriptase peptide variant/class I major histocompatibility complexes.

Designing altered peptide ligands to generate specific immunological reactivity when bound to class I major histocompatibility complexes is important for both therapeutic and prophylactic reasons. We have previously shown that two altered peptides, derived from human immunodeficiency virus (HIV)-reverse transcriptase (RT) residues 309-317, are more immunogenic in vitro than the wild-type peptide. One peptide variant, I1Y, was able to stimulate RT-specific cytotoxic T cells from the blood of three HIV-infected individuals better than the wild-type RT peptide. Both I1Y and I1F peptide variants increase the cell surface half-life of the peptide-class I complex approximately 3-fold over that of the RT peptide but have different immunological activities. These peptides are candidates for the design of vaccines for HIV due to their increased immunogenicity. To understand the basis for the increased cell surface stability compared with wild-type peptide and to understand the differences in T cell recognition between I1Y and I1F, we determined the x-ray crystal structures of the two class I MHC-peptide complexes. These structures indicate that the increased cell surface half-life is due to pi-pi stacking interactions between Trp-167 of HLA-A2.1 and the aromatic P1 residues of I1F and I1Y. Comparison of the structures and modeling potential T cell receptor (TCR) interactions suggests that T cell interactions and immunogenicity are different between I1Y and I1F for two reasons. First, subtle changes in the steric and polar properties of the I1Y peptide affect TCR engagement. Second, water-mediated hydrogen bond interactions between the P1-Tyr and the P4-Glu peptide residues increase peptide side chain rigidity of residues critical for TCR engagement.

Histochemical localization of reverse transcriptase in polytene chromosomes of chironomids.

The localization of a reverse transcriptase-related protein in salivary gland polytene chromosomes was investigated by immunohistochemistry in two species of Chironomus. The antibodies used were raised against a recombinant protein containing phylogenetically conserved motifs of reverse transcriptases and derived from an abundant non-LTR element previously identified in Chironomus. Immunoreactive protein was found in some telomeres, in a centromeric region, in a few interstitial bands and in Balbiani ring 3. The telomeric signal was probably dependent on transcription and increased dramatically when telomeric heat shock puffs were induced. A correlation with transcription was also seen in Balbiani ring 3, the immunobinding of which disappeared after inhibition of transcription with actinomycin D.

Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein.

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.