HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 µM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 µM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

Influence of Pretreatment with Immunosuppressive Drugs on Viral Proliferation.

Immunosuppressive drugs are used to make the body less likely to reject transplanted organs or to treat autoimmune diseases. In this study, five immunosuppressive drugs including two glucocorticoids (dexamethasone and prednisolone), one calcineurin inhibitor (cyclosporin A), one non-steroid anti-inflammatory drug (aspirin), and one antimetabolite (methotrexate) were tested for their effects on viral proliferation using feline foamy virus (FFV). The five drugs had different cytotoxic effects on the Crandell-Ress feline kidney (CRFK) cells, the natural host cell of FFV. Dexamethasone-pretreated CRFK cells were susceptible to FFV infection, but pretreatment with prednisolone, cyclosporin A, aspirin, and methotrexate showed obvious inhibitory effects on FFV proliferation, by reducing viral production to 29.8-83.8% of that of an untreated control. These results were supported by western blot, which detected viral Gag structural protein in the infected cell lysate. As our results showed a correlation between immunosuppressive drugs and susceptibility to viral infections, it is proposed that immune-compromised individuals who are using immune-suppressive drugs may be especially vulnerable to viral infection originated from pets.

Ultrasensitive Immunoassay for Simian Immunodeficiency Virus p27CA.

Although effective for suppressing viral replication, combination antiretroviral treatment (cART) does not represent definitive therapy for HIV infection due to persistence of replication-competent viral reservoirs. The advent of effective cART regimens for simian immunodeficiency virus (SIV)-infected nonhuman primates (NHP) has enabled the development of relevant models for studying viral reservoirs and intervention strategies targeting them. Viral reservoir measurements are crucial for such studies but are problematic. Quantitative polymerase chain reaction (PCR) assays overestimate the size of the replication competent viral reservoir, as not all detected viral genomes are intact. Quantitative viral outgrowth assays measure replication competence, but they suffer from limited precision and dynamic range, and require large numbers of cells. Ex vivo virus induction assays to detect cells harboring inducible virus represent an experimental middle ground, but detection of inducible viral RNA in such assays does not necessarily indicate production of virions, while detection of more immunologically relevant viral proteins, including p27CA, by conventional enzyme-linked immunosorbent assays (ELISA) lacks sensitivity. An ultrasensitive digital SIV Gag p27 assay was developed, which is 100-fold more sensitive than a conventional ELISA. In ex vivo virus induction assays, the quantification of SIV Gag p27 produced by stimulated CD4+ T cells from rhesus macaques receiving cART enabled earlier and more sensitive detection than conventional ELISA-based approaches and was highly correlated with SIV RNA, as measured by quantitative reverse transcription PCR. This ultrasensitive p27 assay provides a new tool to assess ongoing replication and reactivation of infectious virus from reservoirs in SIV-infected NHP.

Development of an antigen-capture ELISA for the detection of the p27-CA protein of HERV-K(HML-2).

The detection or quantification of retroviruses is often achieved using an antigen-capture ELISA (AC-ELISA) that targets the Gag capsid (CA) protein. We report here the development of an AC-ELISA specific for the p27-CA protein of HERV-K(HML-2). A monoclonal p27-specific antibody is used for capture and a polyclonal anti-p27-CA immune serum generated in rabbits serves for detection. The assay was shown to be specific for HERV-K(HML-2), showing no evidence of cross reactivity with the human retroviruses HIV-1, HIV-2 and HTLV-1 or with XMRV (as a model non-human gammaretrovirus). Using purified recombinant antigen, the limit of detection was shown to be 130pg/ml. The AC-ELISA can be used to quantify HERV-K(HML-2) expression in teratocarcinoma cell lines and to normalize HERV particles generated by transfecting HEK 293T cells with full-length molecular clones. This novel AC-ELISA also proved useful in studies of virus regulation, for example in demonstrating that HERV-K(HML-2) expression is dramatically enhanced by overexpression of Staufen-1, a binding partner of the HERV-K(HML-2) Rec protein. This specific and sensitive HERV-K(HML-2) AC-ELISA will be a useful tool for investigating many aspects of endogenous retroviruses, from basic research to the role they may play in human diseases or as a surrogate marker for particular diseases.

Distinct Morphology of Human T-Cell Leukemia Virus Type 1-Like Particles.

The Gag polyprotein is the main retroviral structural protein and is essential for the assembly and release of virus particles. In this study, we have analyzed the morphology and Gag stoichiometry of human T-cell leukemia virus type 1 (HTLV-1)-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, we have observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Other morphological features that have been previously observed with other retroviruses include: (1) a Gag lattice with multiple discontinuities; (2) membrane regions associated with the Gag lattice that exhibited a string of bead-like densities at the inner leaflet; and (3) an arrangement of the Gag lattice resembling a railroad track. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date.

In Situ Staining and Laser Capture Microdissection of Lymph Node Residing SIV Gag-Specific CD8+ T cells--A Tool to Interrogate a Functional Immune Response Ex Vivo.

While a plethora of data describes the essential role of systemic CD8+ T cells in the control of SIV replication little is known about the local in situ CD8+ T cell immune responses against SIV at the intact tissue level, due to technical limitations. In situ staining, using GagCM9 Qdot 655 multimers, were here combined with laser capture microdissection to detect and collect SIV Gag CM9 specific CD8+ T cells in lymph node tissue from SIV infected rhesus macaques. CD8+ T cells from SIV infected and uninfected rhesus macaques were also collected and compared to the SIV GagCM9 specific CD8+ T cells. Illumina bead array and transcriptional analyses were used to assess the transcriptional profiles and the three different CD8+ T cell populations displayed unique transcriptional patterns. This pilot study demonstrates that rapid and specific immunostaining combined with laser capture microdissection in concert with transcriptional profiling may be used to elucidate phenotypic differences between CD8+ T cells in SIV infection. Such technologies may be useful to determine differences in functional activities of HIV/SIV specific T cells.

Proviral loads of human T-lymphotropic virus Type 1 in asymptomatic carriers with different infection routes.

High human T-lymphotropic virus Type 1 (HTLV-1) proviral DNA load (PVL) has been reported to be one risk factor for the development of adult T-cell leukemia/lymphoma (ATL). ATL is also believed to develop in HTLV-1 carriers who acquire infection perinatally. ATL cells have been reported to frequently harbor defective provirus. In our study, PVLs for three different regions of HTLV-1 provirus (5'LTR-gag, gag and pX) were measured in 309 asymptomatic carriers with different infection routes. PVLs for the pX region in 21 asymptomatic carriers with maternal infection was significantly higher than in 24 carriers with spousal infection. Among 161 carriers with relatively high pX PVLs (equal to or greater than 1 copy per 100 peripheral blood mononuclear cells), 26 carriers (16%) had low gag PVL/pX PVL (less than 0.5) and four (2%) had low 5'LTR-gag PVL/pX PVL (less than 0.5). Low gag PVL/pX PVL ratio, which reflects deficiency and/or polymorphism of HTLV-1 proviral DNA sequences for the gag region, was also associated with maternal infection. These data suggest that HTLV-1 carriers with maternal infection tend to have high PVLs, which may be related to provirus with deficiency and/or the polymorphism of proviral DNA sequences. In addition, there is a possibility that this ratio may be used as a tool to differentiate the infection routes of asymptomatic HTLV-1 carriers, which supports the need for a large scale study.

Functional mechanisms of the cellular prion protein (PrP(C)) associated anti-HIV-1 properties.

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.

Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release: relationship with the Gag-to-GagPol ratio.

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stochiometry.

BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells. RESULTS: The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions. CONCLUSIONS: In summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry.

Incomplete IgG response to HIV-1 proteins and low avidity levels in recently converted HIV patients treated with early antiretroviral therapy.

OBJECTIVES: To evaluate the evolution of antibody avidity and Western blot reactivity in recently infected HIV-1 subjects and to study the impact of highly active antiretroviral therapy (HAART) on avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with recent HIV-1 infection. METHODS: Thirty-six HIV-1 seroconverters were enrolled in this study and followed longitudinally over 24 months to evaluate if the administration of antiretroviral therapy during primary infection affects Western blot reactivity and the evolution of antibody avidity. The patients were divided into two groups; group A consisted of 19 HIV-1-untreated patients who did not receive any drug treatment during our follow-up period; group B consisted of 17 subjects who were treated early with an association of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) within 3 months after seroconversion. RESULTS: At diagnosis, Western blot analysis and avidity index (mean value) were exactly matched in untreated and treated patients; subsequently, however, a significantly lower reactivity to HIV-1 pol and gag proteins and a lower avidity index (mean values) were observed in HAART-treated patients up until the end of the follow-up period. CONCLUSIONS: The impaired production and maturation of the humoral immunological response in antiretroviral-treated patients might be related to a rapid suppression of HIV replication, driven by HAART. These results could have important implications in understanding the complex mechanism of the immune response during HIV infection.

Studies on long-term infection of human cells with Porcine endogenous retrovirus.

A major concern in pig-to-human xenotransplantations is the potential risk of transmission of Porcine endogenous retroviruses (PERVs) integrated in the pig genome. Our previous work has shown that PERV provirus genes and gag protein can be detected in human embryonic kidney HEK-293 cells during a long-term infection with PERV (Yu et al., Transplant. Proc. 37, 496-499, 2005). In this study, we continued studying the long-term (>6 months) PERV infection of HEK-293 cells. The results showed no significant differences in morphology, growth, apoptosis, and [(3)H]-thymidine incorporation between PERV-infected and uninfected cells. The PERV LTR sequence showed only an insignifcant mutation after the long-term infection. PERV infection had no effect on the transcription of genes of Human endogenous retrovirus (HERV) naturally occurring in HEK-293 cells. Summing up, this study indicated that a long-term PERV infection of HEK-293 cells in vitro does not result in any significant changes in host cells as well as in PERV LTR sequence.

Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cell-associated and cell-free transmission of the virus are possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines after their exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS.

Amino acid alterations in Gag that confer the ability to grow in simian cells on HIV-1 are located at a narrow CA region.

We previously generated a prototype monkey-tropic human immunodeficiency virus type 1 (HIV-1) designated NL-DT5R. This viral clone has a small region of simian immunodeficiency virus (SIV) within Gag capsid (CA) protein and also SIV Vif protein, but displays a poor growth phenotype in simian cells. To improve the growth potential of NL-DT5R, we have constructed a series of its gag variant viruses. Out of fourteen viral clones generated, five were infectious for simian HSC-F cells, and two of the infectious variants grew similarly with NL-DT5R. Taking their genome structures into consideration, our data here clearly show that a narrow CA region within the Gag protein, i.e., the domain around cyclophilin A (CypA)-binding loop, is critical for the growth ability of HIV-1 in simian cells.

An optimized nested polymerase chain reaction (PCR) approach allows detection and characterization of human immunodeficiency virus type 1 (HIV-1) env and gag genes from clinical samples.

The needs for development and/or improvement of molecular approaches for microorganism detection and characterization such as polymerase chain reaction (PCR) are of high interest due their sensitivity and specificity when compared to traditional microbiological techniques. Considering the worldwide importance of human immunodeficiency virus type 1 (HIV-1) infection, it is essential that such approaches consider the genetic variability of the virus, the heterogeneous nature of the clinical samples, the existence of contaminants and inhibitors, and the consequent needs for standardization in order to guarantee the reproducibility of the methods. In this work we describe a nested PCR assay targeting HIV-1 virus gag and env genes, allowing specific and sensitive diagnosis and further direct characterization of clinical samples. The method described herein was tested on clinical samples and allowed the detection of HIV-1 presence in all samples tested for the gag gene and 90.9% for the env gene, revealing sensitivities of 1 fg and 100 fg, respectively. Also, no cross-reactions were observed with DNA from infected and noninfected patients and the method allowed detection of the env and gag genes on an excess of 10(8) and 10(4) of human deoxyribonucleic acid (DNA), respectively. Furthermore, it was possible to direct sequence all amplified products, which allowed the sub typing of the virus in clinical samples.

IL-15 treatment during acute simian immunodeficiency virus (SIV) infection increases viral set point and accelerates disease progression despite the induction of stronger SIV-specific CD8+ T cell responses.

In this study, we examined the effect of in vivo treatment of acutely SIV-infected Mamu-A*01+ rhesus macaques with IL-15. IL-15 treatment during acute infection increased viral set point by 3 logs and accelerated the development of simian AIDS in two of six animals with one developing early minimal lesion SIV meningoencephalitis. Although IL-15 induced a 2- to 3-fold increase in SIV-specific CD8+ T cell and NK cell numbers at peak viremia and reduced lymph node (LN) SIV-infected cells, this had no impact on peak viremia and did not lower viral set point. At viral set point, however, activated SIV-specific CD8+ T cells and NK cells were reduced in the blood of IL-15-treated animals and LN SIV-infected cells were increased. Week 30 LN from IL-15-treated animals had significantly increased Gag-specific CD8+ T cell numbers, whereas total cell, lymphocyte, and CD4+ T cell numbers were reduced. IL-15 treatment significantly reduced anti-SIV Ab concentrations at week 3 and viral set point. IL-15 increased Ki-67+CD4+ T cells at week 1 of treatment and reduced blood CCR5+ and CD45RA-CD62L- CD4+ T cells. The frequency of day 7 Ki-67+CD4+ T cells strongly correlated with viral set point. These findings suggest that CD4+ T cell activation during acute infection determines subsequent viral set point and IL-15 treatment by increasing such activation elevates viral set point. Finally, IL-15-treated acutely SIV-infected primates may serve as a useful model to investigate the poorly understood mechanisms that control viral set point and disease progression in HIV infection.

HIV-1 buds and accumulates in "nonacidic" endosomes of macrophages.

Macrophages represent viral reservoirs in HIV-1-infected patients and accumulate viral particles within an endosomal compartment where they remain infectious for long periods of time. To determine how HIV-1 survives in endocytic compartments that become highly acidic and proteolytic and to study the nature of these virus-containing compartments, we carried out an ultrastructural study on HIV-1-infected primary macrophages. The endosomal compartments contain newly formed virions rather than internalized ones. In contrast to endocytic compartments free of viral proteins within the same infected cells, the virus containing compartments do not acidify. The lack of acidification is associated with an inability to recruit the proton pump vacuolar ATPase into the viral assembly compartment. This may prevent its fusion with lysosomes, since acidification is required for the maturation of endosomes. Thus, HIV-1 has developed a strategy for survival within infected macrophages involving prevention of acidification within a devoted endocytic virus assembly compartment.

The location in cartilage of infectious retrovirus in cats infected with feline leukemia virus.

BACKGROUND: Previous studies have suggested that articular cartilage allografts were not likely to transmit infectious retrovirus since viral DNA could not be isolated from chondrocytes of infected individuals. However, the ability of the extracellular matrix of articular cartilage to harbor and transmit a retrovirus has not been examined. We hypothesized that articular cartilage fragments, but not isolated chondrocytes, from cats systemically infected with feline leukemia virus (FeLV) are capable of transmitting infectious retrovirus. METHODS: Fresh cartilage segments and chondrocytes isolated from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cocultured with segments of cartilage, isolated chondrocytes, or fragments of cortical bone from each infected cat. The FeLV p27 antigen was measured in the coculture media by enzyme-linked immunosorbent assay. In addition, FeLV proviral nucleic acids were quantified by real-time quantitative polymerase chain reaction with use of DNA extracted from feline embryonic fibroblast cell cocultures as well as isolated chondrocytes. Immunohistochemistry was used to assess for FeLV p27 antigen in both intact cartilage fragments and isolated chondrocytes. RESULTS: Feline embryonic fibroblast cells cocultured with cartilage fragments from each of the five FeLV-infected cats all demonstrated high levels of proviral DNA, indicating transmission of infective virus. In addition, media from all cocultures of feline embryonic fibroblast cells and chondral fragments became positive for p27 antigen, indicating active viral replication. In contrast, cocultures of feline embryonic fibroblast cells and isolated chondrocytes from all FeLV-infected cats were negative for proviral DNA and p27 antigen. Likewise, no proviral nucleic acids could be detected in isolated chondrocytes from any infected cats. Cocultures of feline embryonic fibroblast cells with cortical bone fragments were positive for proviral DNA and p27 antigen. Immunohistochemical staining of cartilage fragments from FeLV-infected cats demonstrated the presence of p27 antigen throughout the extracellular matrix, but the p27 antigen was not detected in isolated chondrocytes. CONCLUSIONS: Articular cartilage fragments can readily transmit infectious retrovirus, but isolated chondrocytes were likely not the source of the infectious virus because they did not harbor proviral DNA or p27 antigen.

Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis.

Clostridium perfringens (C. perfringens) has been developed as a potential oral delivery vehicle to deliver antigens or therapeutic compounds to Gut-Associated Lymphoid Tissue (GALT). However, this recombinant C. perfringens carries a plasmid-encoded expression system, which raises several safety concerns regarding possible horizontal plasmid transfer and spread of plasmid-associated antibiotic resistant genes. Furthermore, this bacterium produces the extracellular theta toxin, which poses a potential safety issue for general administration. Using a Clostridium-specific-targetron donor plasmid, we inserted the Simian Immunodefiency Virus (SIV) p27 gene into the theta toxin gene (pfoA) on the C. perfringens chromosome, which simultaneously inactivated the theta gene and introduced SIV p27 gene onto the bacterial chromosome. Such mutant C. perfringens without an input plasmid or antibiotic resistant gene stably produced a large amount of SIV p27 protein during sporulation and did not produce theta toxin. Upon oral feeding of the mutant bacteria to mice, intact p27 protein was detected in the lower GI tract. The re-engineered C. perfringens provides a biosafe efficient oral vehicle to deliver antigen to the gastrointestinal tract.

Isolation and characterization of mouse-human microcell hybrid cell clones permissive for infectious HIV particle release.

Mouse cells are non-permissive to human immunodeficiency virus type 1 (HIV) in that there is a pronounced post-integration block to viral replication. We have recently demonstrated that mouse-human somatic cell hybrids that contain human chromosome 2 increase both HIV Capsid (CA) production and infectious virus release. Here we report on the isolation of three mouse-human microcell hybrids (MCHs) that behave similarly, starting from a pool of 500 MCH clones. Release of virus was specific to HIV and cell revertants that no longer contained any human chromosome fragments did not release CA or infectious virus. Two of the three cell clones were identical as judged by PCR STS content and fluorescence in situ hybridization (FISH) and contained a single 2-12 human chromosome chimera. The third cell clone only contained human chromosome 12, as determined by PCR, FISH, and microarray analyses. There were no consistent differences in Gag protein and spliced/unspliced viral RNA levels between mouse cell lines. CMV promoter-driven, codon-optimized gag-pol had no effect on infectious HIV release from these mouse cells, despite allowing Gag targeting and increasing CA production. These permissive mouse-human MCHs and their corresponding non-permissive revertants may prove useful for mechanistic studies and also for identifying the responsible gene(s) or factor(s) involved in the production of HIV.