Retrovirus-like Particles Research Articles

Murine leukemia virus clearance by flocculation and microfiltration

Clearance of murine leukemia virus from CHO cell suspensions by flocculation and microfiltration was investigated. Murine leukemia virus is a retrovirus that is recommended by the U.S. Food and Drug Administration for validating clearance of retrovirus-like particles. Due to biosafety considerations, an amphotropic murine leukemia virus vector (A-MLV) that is incapable of self-replication was used. Further, A-MLV is incapable of infecting CHO cells, thus ensuring that infection of the CHO cells in the feed did not result in a reduced virus titer in the permeate. The virus vector contains the gene for the enhanced green fluorescent protein (EGFP) to facilitate assaying for infectious virus particles. The virus particles are 80-130 nm in size. The feed streams were flocculated using a cationic polyelectrolyte. Microfiltration was conducted using 0.1 and 0.65 microm pore size hollow fiber membranes. The level of virus clearance in the permeate was determined. For the 0.1 microm pore size membranes a 1,000-fold reduction in the virus titer in the permeate was observed for feed streams consisting of A-MLV, A-MLV plus flocculant, A-MLV plus CHO cells, and A-MLV plus flocculant and CHO cells. While the flocculant had little effect on the level of virus clearance in the permeate for 0.1 microm pore size membranes, it did lead to higher permeate fluxes for the CHO cell feed streams. Virus clearance experiments conducted with 0.65 microm pore size membranes indicate little clearance of A-MLV from the permeate in the absence of flocculant. However, in the presence of flocculant the level of virus clearance in the permeate was similar to that observed for 0.1 microm pore size membranes. The results obtained here indicate that significant clearance of A-MLV is possible during tangential flow microfiltration. Addition of a flocculant is essential if the membrane pore size is greater than the diameter of the virus particles. Flocculation of the feed stream leads to an increase in the permeate flux.

A critique of the Montagnier evidence for the HIV/AIDS hypothesis

In 1983 Luc Montagnier and his colleagues claimed to have discovered a novel retrovirus presently known as human immunodeficiency virus (HIV). By 1984 HIV was almost universally accepted to be the cause of AIDS. However, 20 years later, HIV cannot account for the phenomena for which the retroviral hypothesis was proposed, namely, Kaposi's sarcoma, decrease in T4 lymphocytes and thus the opportunistic infections in AIDS patients which were assumed to be the direct results of this decrease. Agents other than HIV to which patients belonging to the AIDS risk groups are exposed cause decrease in T4 cells. Neither have the main predictions of the HIV hypothesis been fulfilled. HIV seropositivity in the developed countries still remains restricted to the original high risk groups, no HIV vaccine exists, and no successful animal model has been developed. In this communication, we critically analyse the evidence which in 1983 was claimed to prove the existence of HIV. The phenomena which Montagnier and his colleagues considered proof for the existence of HIV are detection of reverse transcriptase activity; the presence of retrovirus-like particles in the culture; immunological reactivity between proteins from the culture supernatant which, in sucrose density gradients, banded at the density of 1.16 g/ml (“purified virus”) and antibodies in a patient's (BRU) serum. Reverse transcriptase activity can be found in viruses other than retroviruses and in all normal cells. Reverse transcription can be brought about not only by the enzyme reverse transcriptase but also by normal, cellular DNA polymerases. Retrovirus-like particles are ubiquitous in cultures not infected with retroviruses, especially in conditions employed by Montagnier et al. From the reaction between proteins in the “purified virus” and antibodies in the patient serum Montagnier concluded that the proteins were HIV proteins and the antibodies were HIV antibodies. Since all antibodies are polyspecific, from such a reaction it is not possible to define the origin of even one reactant let alone both. Even if this were possible, since Montagnier's “purified virus” did not contain particles with the “morphology typical of retroviruses”, the proteins cannot be retroviral. We conclude that, these phenomena are non-specific to retroviruses and thus cannot be considered proof for the existence of a unique retrovirus HIV.

Monoclonal antibody 667 recognizes the variable region A motif of the ecotropic retrovirus CasBrE envelope glycoprotein and inhibits Env binding to the viral receptor.

Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D(57), is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

Semliki Forest virus vectors for gene therapy

Semliki Forest virus (SFV) vectors transduce a broad range of mammalian and non-mammalian cells, generating high levels of transient expression of heterologous proteins. Generally, they induce apoptosis in mammalian host cells, leading to rapid cell death. These features have made SFV attractive for various gene therapy applications. Recombinant particles, naked RNA and plasmid DNA containing SFV replicons, demonstrate a strong immune response against recombinantly expressed proteins, which has shown protection against tumour challenges. Intratumoural injection of SFV particles has resulted in tumour regression. SFV vectors have been used for production of retrovirus-like particles. Recently, encapsulation of SFV particles into liposomes has generated highly efficient targeting to tumours. Novel SFV vectors based on point mutations in the non-structural genes, and avirulent SFV strains, have further widened the application range.

Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes.

A single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal ("Psi") in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and Psi- particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed.

Analysis of bovine leukemia virus gag membrane targeting and late domain function.

Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain).

Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells.

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.

Alphavirus vectors as tools in cancer gene therapy.

Alphavirus vectors, particularly those based on the replicon of Semliki Forest virus, have shown great potential as gene delivery vehicles for various applications in cancer gene therapy. The rapid production of high-titer recombinant SFV particles, which show impressive transduction rates in various mammalian cell lines, primary cultures and in vivo, results in high levels of transgene expression. Additionally, SFV vectors induce apoptosis in transduced host cells, which can further increase their efficiency in tumor therapy. Because of the broad host range some attempts to target the gene delivery have been engineered for Sindbis virus vectors, where IgG binding domains of protein A have been introduced into the envelope structure of the recombinant particles to allow attachment of virus to host cells through the interaction of protein A with monoclonal antibodies. SFV vectors have also been employed for the production of retrovirus-like particles for establishment of long-term gene expression. Tumor vaccine approaches have been taken by injection of SFV vectors as naked RNA molecules, DNA plasmids or recombinant particles to achieve both therapeutic and prophylactic efficacy. The continuous improvement of alphavirus vectors will further expand the application range in the future.

Retrotransposon mdg3 is transferred between somatic cells of unrelated species in coculture

Since retrovirus-like particles of gypsy (mdg4) are capable of interspecific transfer, other Drosophila melanogaster gypsy-related retrotransposons were tested for this property. As a donor and a recipient, D. melanogaster and D. virilis cultured cells were used. Recipient cell DNA was analyzed with probes directed to mdg1, mdg3, 17.6, 297, 412, or B104/roo. Transfer was demonstrated for mdg3, which lacks env. The possible mechanism of transfer is discussed.

Tracking the assembly pathway of human immunodeficiency virus type 1 Gag deletion mutants by immunogold labeling.

The Pr55gag gene product of human immunodeficiency virus type 1 (HIV-1) is sufficient to direct the formation of retrovirus-like particles (RVLPs). Recent biochemical evidence has indicated the presence of Gag intermediates in the cytoplasm; however, the Gag assembly process into RVLPs remains incompletely defined. The authors present here the subcellular localization of Gag mutant proteins in BSC40 and Jurkat cells by immunoelectron microscopy (IEM). The full Gag/Pol and Gag precursors, a C-terminal deletion mutant lacking a portion of nucleocapsid (NC), and all p6Gag gave rise to similar levels of RVLPs at the cell surface. A C-terminal deletion of all NC and p6Gag abrogated particle formation, whereas p24 was found in patches at the cell surface. Deletion of matrix (MA) sequences from Gag resulted in intracellular particles, and myristylation was not required for particle formation in the context of the MA deletion. Matrix expression was enhanced with Gag/Pol or Env coexpression as determined by semiquantitative IEM. p24 protein was targeted at vacuolar and mitochondrial membranes, but not at Golgi cisternae. In addition, aggregations of Gag intermediates and RVLPs in the cytoplasm, rough endoplasmic reticulum, cisternae, and mitochondria were noted. These results provide defined in situ evidence that HIV-1 particle assembly occurs in the cytosol in addition to budding at most intracellular membranes.

VSV-G envelope glycoprotein forms complexes with plasmid DNA and MLV retrovirus-like particles in cell-free conditions and enhances DNA transfection.

We have previously shown that vesicles containing the spike glycoprotein of the vesicular stomatitis virus (VSV-G) can associate efficiently with immature, non-infectious, envelope-deficient retrovirus-like particles assembled by packaging cells to produce infectious, pseudotyped viruses in cell-free conditions in vitro. We have also previously reported that VSV-G can enhance DNA lipofection efficiency by interacting with liposomes to form fusogenic, serum-stable liposomes with enhanced transfection properties. Here, we report that VSV-G can form a complex directly with naked plasmid DNA in the absence of a lipofection reagent and can thereby enhance the transfection efficiency of the naked plasmid vector. Sucrose gradient sedimentation analysis demonstrated that VSV-G can also associate with plasmid DNA and murine leukemia virus (MLV) gag-pol particles to form ternary complexes that co-sediment with high DNA transfecting activity. The increased transfection efficiency with VSV-G was dependent on the presence of the polycation (Polybrene) in the culture medium during transfection. Enhanced transfection was abolished by a neutralizing antibody to VSV-G. These results may be useful in the study of retrovirus assembly, in the further design of hybrid DNA-based retrovirus-like vectors, and in the full in vitro, cell-free assembly of infectious virus-like particles from component parts.

Characterization of a hormonally induced reverse transcriptase (RT) from the human breast cancer cell line T47D: a possible involvement in human breast cancer

Since the discovery of the mouse mammary tumor virus (MMTV), which was shown to be involved in mouse mammary carcinoma, there has been an attempt to discover similar viruses associated with human breast cancer. We have already shown that the human mammary carcinoma cell line T47D releases retrovirus-like particles in response to steroid treatment. An RT transcript from T47D cells was isolated, using RT-PCR with primers based on the published T47D endogenous retroviral pol sequences. The PCR product encodes a 372-amino-acid long protein. The new T47D RT is almost identical to both previously described RTs from T47D cells, as well as to the enzymatically active RT from human bone marrow cells (95 and 97% identity, respectively). The DNA product was cloned into a bacterial expression system. A 42-kDa RT-related fragment was expressed, purified, and used to immunize rabbits. The antibodies recognize a 60-70 kDa hormonally induced protein specifically in T47D cells. Moreover, steroid hormones induce the apperance of RT protein foci in the cell cytoplasm, as demonstrated by confocal laser microscopy. A parallel hormonal induction of the RT activity in cell supernatants was observed. Expression of the RT-related protein was also detected in tumor cells of breast cancer biopsies sections. These results support the idea that retroviruses may be associated with human breast carcinoma.

HIV-1 Penetrates Coronary Artery Endothelial Cells by Transcytosis

The pathogenesis of HIV-1-related cardiomyopathy is poorly understood, but HIV-1 has been detected in cardiomyocytes. Whether HIV-1 penetrates into the myocardium by infection of coronary artery endothelial cells (CAEC) or using transcellular or paracellular routes across CAEC has not been resolved. A model of the CAEC barrier was constructed with primary CAEC (derived from human coronary vessels). Polymerase chain reaction (PCR) assay, infectious assay, and immunofluorescence were employed to show abortive nature of HIV-1 infection of CAEC. Tight junction (TJ) and cell adhesion proteins were visualized by immunofluorescence. The time course of HIV-1 invasion was measured by HIV-1 RNA assay. Inulin permeability assay determined paracellular leakage. Transmission electron microscopy demonstrated virus-induced endothelial vacuolization. Despite a strong display on CAEC of CXCR4 and a lesser expression of CCR3 and CCR5, HIV-1 did not productively replicate in CAEC, as shown by infectious assay, immunofluorescence, and electron microscopy. HIV-1 infection of CAEC was abortive with minimal reverse transcription of strong stop DNA and pol but not full-length or two LTR DNA circles. Upon infection of the model with 1 million RNA copies of HIV-1JR-FL, virus penetration 2 hr postinfection (PI) was negligible but increased by 1,750% 24 hr PI. The paracellular permeability increased during this period by only 25%. Neither AOP-RANTES nor v-MIPII significantly reduced HIV-1JR-FL invasion. Virus infection did not alter the integral TJ protein occludin and the TJ-associated protein ZO-1. HIV-1 exposed CAEC and brain microvascular endothelial cells (BMVEC) developed extensive cytoplasmic vacuolization with retroviral-like particles in the vacuoles. The endothelium is not an impenetrable barrier to HIV-1. The virus opens a transcellular route across coronary and brain endothelia in cytoplasmic vacuoles.

Phenotypic heterogeneity of human endogenous retrovirus particles produced by teratocarcinoma cell lines.

Human endogenous retrovirus (HERV) sequences represent about 0.5% of the human genome. The only HERV known to express virus particles is human teratocarcinoma-derived virus (HTDV), which is now termed HTDV/HERV-K. Between 25 and 50 different copies of HERV-K are present in the human genome, three of which contain full-length genes for viral structural proteins. To determine whether genes of different HERV-K proviruses can be expressed, the morphologies and protein expression patterns of HTDV/HERV-K produced by various human teratocarcinoma cell lines were compared. Three different types of retrovirus-like particles were observed, showing differences in the presence of viral surface proteins and the existence of free mature virions. These distinct morphological features between virion types were in accordance with the results of immunoblotting analyses that revealed differences in the cleavage of a viral Gag protein precursor and the presence of a putative Env protein. These data suggest that different HERV-K proviruses are transcribed in human teratocarcinoma cell lines.

Real-time Quantitative PCR for Retrovirus-like Particle Quantification in CHO Cell Culture

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1×106particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3–6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5′→3′ exonuclease activity of Taq DNA polymerase and utilizes the PRISM™ 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system.

Mapping and characterization of the N-terminal I domain of human immunodeficiency virus type 1 Pr55(Gag).

Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55(Gag) molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55(Gag). The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.

Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain.

The human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55(gag) by itself is capable of assembling into retrovirus-like particles (VLP). In the present study, we attempted to identify the minimal Gag sequences required for the formation of VLP. Our results show that about 80% of Pr55(gag) can be either deleted or replaced by heterologous sequences without significantly compromising VLP production. The smallest chimeric molecule still able to efficiently form VLP was only about 16 kDa. This minimal Gag construct contained the leucine zipper domain of the yeast transcription factor GCN4 to substitute for the assembly function of nucleocapsid (NC), followed by a P-P-P-P-Y motif to provide late budding (L) domain function, and retained only the myristylation signal and the C-terminal capsid-p2 domain of Pr55(gag). We also show that the L domain function of HIV-1 p6(gag) is not dependent on the presence of an active viral protease and that the NC domain of Pr55(gag) is dispensable for the incorporation of Vpr into VLP.

Ultrastructural and karyotypic examination of in vitro produced bovine embryos developed in the sheep uterus

This study examined whether development of bovine in vitro produced (IVP) blastocysts in the sheep uterus resulted in morphologically and karyotypically normal elongation stage bovine blastocysts. Seven day IVP bovine blastocysts, resulting from either in vitro maturation and fertilization, nuclear transfer (NT), or parthenogenic activation, were surgically transferred at the blastocyst stage into sheep uteri. Sheep were sacrificed after 7–9 days, and blastocysts were flushed from their uteri. One of each kind of IVP bovine blastocyst was recovered from sheep uteri for analysis by transmission electron microscopy, and nine NT blastocysts were used to establish cell cultures that were analysed for chromosome complement. TEM analysis of in vivo-derived elongation stage bovine and ovine blastocysts was done for comparative purposes. Most ultrastructural features of the 13–19 day blastocysts were similar to earlier stage blastocysts except that distinct alternative mitochondrial morphologies were found between epiblast and trophectoderm cells. Monociliated cells, presumably nodal cells, were observed in the bovine epiblast and hypoblast, and retrovirus-like particles were elaborated by cells in these same areas. Development in the sheep uterus of IVP bovine blastocysts resulted in the presence of crystalloid bodies in the trophectoderm cells, and apoptotic and necrotic cells were observed in the epiblast tissue. Thus, in vivo incubation in the sheep uterus allowed nearly normal development to the elongated blastocyst stage and may be useful for assessment of NT bovine blastocyst developmental competence. Cell cultures derived from the NT blastocysts had normal chromosome complements suggesting that activation by ionomycin and 6-dimethyl-aminopurine did not cause detrimental changes in ploidy in those blastocysts that developed.

Drosophila tRNAs hyperprocessed in vitro by ribonuclease P.

In transposon copia-related retrovirus-like particles of Drosophila, a 5' half fragment produced by the cleavage of mature initiator methionine tRNA is used as the primer for minus-strand reverse transcription. This cleavage is called hyperprocessing. We have previously reported that the catalytic RNA subunit of RNase P catalyzes this hyperprocessing in vitro and that this cleavage is dependent on the occurrence of an altered conformation of the tRNA substrate. Here, we found that other mature tRNAs of Drosophila were also hyperprocessed by M1 RNA in vitro and that some of such tRNAs were probably alanine and histidine tRNAs. Here we report these two tRNAs can also adopt their alternative conformations very similar to that of initiator methionine tRNA.

Quantitative competitive reverse transcription-PCR as a method to evaluate retrovirus removal during chromatography procedures

Chinese hamster ovary cells used for pharmaceutical protein production express non-infectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing process to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for validation studies. Some chromatography procedures used for pharmaceutical protein purification utilize low pH (<pH 4.0) elution buffers which readily inactivate X-MuLV. Therefore, cell-based infectivity assays are unable to evaluate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by chromatography, a quantitative competitive reverse transcription PCR method capable of quantifying both infectious and non-infectious X-MuLV has been developed. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amount of X-MuLV pRNA in the load pool and the product-containing elution pool represents the extent of X-MuLV removal. This method is an extremely powerful complement to cell based-infectivity assays as it allows physical removal of X-MuLV by chromatography and filtration procedures to be distinguished from X-MuLV inactivation when buffers with the ability to inactivate retrovirus are used.