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PubMed1998

In vitro analysis of AcrySof intraocular lens glistenings in AcryPak and Wagon Wheel packaging.

PURPOSE: To compare the formation of "glistenings" in acrylic intraocular lenses (IOLs) packaged in Wagon Wheel and AcryPak packaging systems. SETTING: John Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. METHODS: Acrylic IOLs in Wagon Wheel (WW) and AcryPak (AP) packaging were analyzed in vitro. The lenses were placed in a test chamber with a controlled temperature and evaluated every 48 hours for 336 hours. The presence of glistenings within the lens optic was assessed using slitlamp evaluation and a quantitative microscopic evaluation with an image analysis system. RESULTS: When maintained at a constant temperature, the WW-packaged IOLs showed no glistening formation and the AP-packaged IOLs, significant glistening formation. Glistenings were noted in the WW-packaged IOLs only under fluctuating temperature conditions. Quantitative image analysis confirmed the presence of glistenings only in AP-packaged IOLs maintained at a constant temperature. CONCLUSION: The glistenings in AcrySof IOLs were temperature dependent and confined to IOLs packaged in AcryPak folders and maintained at constant (body) temperatures. These findings are believed to be consistent with fluid formation within the acrylic optic, somehow related to the AcryPak packaging system.

Acrylates↗
PubMed2002

Active and intelligent packaging: applications and regulatory aspects.

Changes in the way foods are produced, distributed, stored and retailed, reflecting the continuing increase in consumer demands for improved quality and extended shelf-life for packaged foods, are placing ever-greater demands on the performance of food packaging. Consumers want to be assured that the packaging is fulfilling its function of protecting the integrity, quality, freshness and safety of foods. To provide this assurance and help improve the performance of the packaging, innovative active and intelligent packaging concepts are being developed and applied in various countries. In Europe, however, the development and application of active and intelligent packaging systems have been limited thus far. The main reasons are legislative restrictions and a lack of knowledge about consumer acceptance, the efficacy of such systems, and the economic and environmental impact they may have. Therefore, in 1999, a European study was started within the framework of the EU FAIR R&D programme. It aims to initiate amendments to European legislation for food-contact materials to establish and implement active and intelligent systems within the current relevant regulations for packaged food in Europe. This paper presents an overview of existing active and intelligent systems and their current and future food-related applications. In addition, developments and trends in active and intelligent food packaging are discussed. The objectives and the work programme of the European project are reviewed and the results obtained so far are presented. The benefits for both the European consumer and the European food and food-packaging industries are highlighted.

Europe↗
PubMed2005

Migration of contaminants by gas phase transfer from carton board and corrugated board box secondary packaging into foods.

The gas phase transfer of substances from carton board (CB) and corrugated box board (CBB) through intervening layers to foods was studied. Substances covering a boiling point range of 252-425 degrees C and a range of polarities were incorporated into CB and CBB secondary packaging. Benzophenone was present in some CB materials. Where it was not already present in CB or CBB secondary packaging, it was deliberately incorporated for transfer studies. Transfer of substances was measured in nine foodstuff types stored in the secondary packaging at ambient and sub-ambient temperature. The foods were packaged in primary packaging materials that would be used in retail. Two food types were packed and stored in both single- and multipack formats. Foods were sampled at 0, 10, 30, 90 and 200 days and analysed by gas chromatography-mass spectrometry after high-performance size exclusion chromatography clean-up. Percentage transfer was between 0 and 100%. The overall trends were increased transfer of substances with increased storage time; a more rapid transfer of the more volatile substances compared with the less volatile ones and higher levels of transfer of the more volatile substances. No transfer of diheptyl phthalate (DHP) (bp 425 degrees C) as an incorporated substance was detected to any foods over the test period. The presence of an additional layer of packaging (multipack versus single pack) was shown to reduce transfer up to fourfold over 200 days and to increase the lag period for transfer. In terms of slowing transfer, metallized PP/PP laminate proved a more effective barrier than PP which was more effective than paper. It is postulated that there is a cut-off threshold for transfer at ambient and sub-ambient temperatures. Substances that are less volatile than the cut-off are anticipated not to transfer from secondary packaging to foods stored for up to 200 days, where the substances are present in the packaging at or below the levels tested in this study (up to 1 mg dm-2). In this study the volatility cut-off threshold lay between that of 2,2-dimethoxyphenylacetophenone (2,2-DMPAP) (an incorporated substance with bp 352 degrees C) and DHP. Ideally, the cut-off threshold should be expressed in terms of vapour pressure in the packaging material. In practical terms, it may be more appropriate to express as partition coefficient as this is simpler to determine experimentally.

Acetophenones↗
PubMed2002

Microbial control by packaging: a review.

Since early man first used a variety of natural containers to store and eat foods, significant developments in food packaging materials have provided the means to suppress microbial growth as well as protect foods from external microbial contamination. Throughout this progression, packaging materials have been developed specifically to prevent the deterioration of foods resulting from exposure to air, moisture, or pH changes associated with the food or the surrounding atmosphere. Both flexible and rigid packaging materials, alone or in combination with other preservation methods, have been developed to offer the necessary barrier, inactivation, and containment properties required for successful food packaging. Examples of flexible packaging used to inactivate microorganisms associated with foods include controlled atmosphere, vacuum, modified atmosphere, active, and edible packaging. Additionally, the combination of rigid packaging materials made from metal, glass, or plastic with heat provides the most effective and widely used method for inactivating microorganisms. As with all food products, it is necessary to integrate a HACCP-based program to assure quality throughout the packaging operation. In addition to packaging improvements, other novel technologies include the development of detectors for oxygen levels, bacterial toxins, and microbial growth, or the integration of time-temperature indicators for detection of improper handling or storage.

Carbon Dioxide↗
PubMed2005

Practical and dosimetric implications of a new type of packaging for radiographic film.

Recently, Kodak introduced new light-tight packages (vacuum packaging, aluminium layer under black polyethylene and different paper) for their oncology films (EDR-2, X-Omat V and PPL-2). In order to avoid additional uncertainty and to ensure transferability of previously published results, we assessed in this study the effect of the old and new packages on the dosimetric response of EDR-2 radiographic film. Therefore, sensitometric measurements were performed for different film assemblies (new envelope + new paper, old envelope + old paper, new envelope without paper and old envelope without paper). In addition, to assess possible effects of the package on the film depth-dose response, packaged films were irradiated in parallel geometry, and central depth-dose curves were retrieved. For the perpendicular geometry, on the other hand, the effect of the package was assessed at large depth for a high intensity-modulated inverse-pyramid beam. The results of the sensitometric measurements reveal no difference between the packages. However, the white colour of the paper in both the packages induces a dose-dependent increase in optical density (0-0.12) of the film. The depth-dose curves show better reproducibility for the new package and the new paper improves the accuracy of film dosimetry, but despite the company's effort to evacuate the air out of the new envelope, it remains necessary to clamp the films in the phantom for the parallel irradiation geometry. At 5 cm depth, the films irradiated in parallel geometry show an under-response of 3-5% compared to films irradiated perpendicularly. Finally, even at locations of large photon scatter, no filtration effect from the aluminium layer incorporated in the new envelope has been observed for perpendicular irradiation geometry.

Dose-Response Relationship, Radiation↗
PubMed2007

Formation of a multiple protein complex on the adenovirus packaging sequence by the IVa2 protein.

During adenovirus virion assembly, the packaging sequence mediates the encapsidation of the viral genome. This sequence is composed of seven functional units, termed A repeats. Recent evidence suggests that the adenovirus IVa2 protein binds the packaging sequence and is involved in packaging of the genome. Study of the IVa2-packaging sequence interaction has been hindered by difficulty in purifying the protein produced in virus-infected cells or by recombinant techniques. We report the first purification of a recombinant untagged version of the adenovirus IVa2 protein and characterize its binding to the packaging sequence in vitro. Our data indicate that there is more than one IVa2 binding site within the packaging sequence and that IVa2 binding to DNA requires the A-repeat consensus, 5'-TTTG-(N(8))-CG-3'. Furthermore, we present evidence that IVa2 forms a multimeric complex on the packaging sequence. These data support a model in which adenovirus DNA packaging occurs via the formation of a IVa2 multiprotein complex on the packaging sequence.

Adenoviridae↗
PubMed2004

Management of packaging waste in Poland--development agenda and accession to the EU.

In recent years the issue of the municipal waste in Poland has become increasingly topical, with a considerable rise in the waste generation, much of which can be attributed to a boom in product packaging (mainly plastic). The annual production of plastics packaging has been constantly increasing over the last 20 to 30 years, and now exceeds 3.7 million tons. Due to a lack of processing technologies and poorly developed selective segregation system, packaging waste is still treated as a part of the municipal solid waste (MSW) stream, most of which is landfilled. As a result of Poland's access to the European Union, previous legal regulations governing municipal waste management have been harmonized with those binding on the member countries. One of the main changes, the most revolutionary one, is to make entrepreneurs liable for environmental risks resulting from the introduction of packaging to the market, and for its recycling. In practice, all entrepreneurs are to ensure recovery, and recycling, of used packaging from products introduced to the market at the required level. In recent year, the required recycling levels were fulfilled for all types of materials but mainly by large institutions using grouped and transport packaging waste for that matter. Household packaging gathered in the selective segregation system at the municipalities was practically left alone. This paper is an attempt to describe the system and assess the first year of functioning of the new, revamped system of packaging waste management in Poland. Recommendations are made relating to those features that need to be included in packaging waste management systems in order to maximize their sustainability and harmonization with the EU legal system.

Conservation of Natural Resources↗
PubMed1998

A nondestructive leak detection method for flexible food packages using hydrogen as a tracer gas.

A nondestructive leak detection method developed at Technical Research Centre of Finland (VTT) was tested for both gas-flushed and vacuum flexible packages. In the method, a gas package containing 0.5 to 5.0% (vol/vol) hydrogen in nitrogen was positioned in a test chamber, a controlled vacuum was pulled in the chamber through a pipe connected to a hydrogen sensor, and leaking packages were detected by the sensor as increased H2 concentration. The H2 tracer gas (0.5 to 5.0%) was introduced into leaking finished vacuum packages at 200 kPa pressure. Within 1 to 4 s the developed test method was able to detect leaks down to 10 to 15 microns and 20 to 30 microns in diameter in commercially manufactured gas-flushed packages filled with roasted meat balls and vacuum packages filled with ground coffee, respectively. Before leak testing, the vacuum packages were charged with H2 for 30 s. The sensitivity and leak detection time of the test method were improved when the H2 concentration in the package was increased and when the free space in the test chamber was decreased. The evaluated H2 concentrations did not affect the sensory or microbiological quality of the roasted meat balls. This study clearly demonstrated that the hydrogen tracer gas leak detection method has potential to be further developed as a fast, nondestructive, on-line leak testing apparatus for flexible packages with or without a headspace.

Animals↗
PubMed1990

Analysis of retroviral packaging lines for generation of replication-competent virus.

Amplification of retroviral vector sequences occurs in cocultures of ecotropic and amphotropic packaging cell lines. Mixed packaging line cocultures were used to determine both the host range and time of appearance of replication-competent virus after introduction of a retroviral vector. Replication-competent virus was generated at a characteristic time for a given ecotropic and amphotropic packaging line combination. The time required to generate replication-competent virus in a packaging line varied with the number of recombination events necessary to generate replication-competent virus from the retroviral sequences present in the line. The psi 2 packaging line generated replication-competent virus within 10 days after transfection of the N2 vector into a mixture of psi 2 (ecotropic) and PA317 (amphotropic) packaging cells. Under the same conditions, it took only 3 days to develop replication-competent virus in psi 2/PA12 cocultures. The host range of replication-competent virus was used to identify the packaging line that initially generates virus. Each packaging line combination generated replication-competent virus at a characteristic time and this time period can be used as a measure of the "safety" of the packaging line and vector combination.

Animals↗
PubMed1994

Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis.

Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gp18 and gp19. We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate. gp19 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by site-directed mutagenesis. gp19 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging activity in the defined and crude systems: group 1 mutants were defective in both systems (gp19-G61D, which is a gp19 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group 4 mutants had almost the same activity as gp19-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.

Adenosine Triphosphatases↗
PubMed2001

Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers.

Recently developed helper virus-free methods of herpes simplex virus (HSV) amplicon vector packaging provide stocks that are virtually devoid of the cytotoxic component normally associated with traditional helper virus-based packaging methods. These approaches involve cotransfection of amplicon plasmid DNA with either a five-cosmid set or a bacterial artificial chromosome (BAC) that contains the HSV genome without its cognate pac signals. Helper virus-free amplicon packaging produces low-titer stocks (<10(5) expressing particles/ml) that exhibit a high frequency of pseudotransduction. In an effort to enhance amplicon titers, we introduced in trans a genomic copy of the virion host shutoff (vhs) protein-encoding gene UL41 into both cosmid- and BAC-based packaging strategies. Cotransfection of this plasmid with the amplicon and packaging reagents results in a 10-fold higher amplicon titer, and stocks that do not exhibit the pseudotransduction phenomenon. To further enhance packaging efficiency, the HSV transcriptional activator VP16 was introduced into packaging cells 1 day before the packaging components. Pre-loading of packaging cells with VP16 led to an additional enhancement of amplicon titers, an effect that did not occur in the absence of vhs. Increased helper virus-free amplicon titers resulting from these modifications will make in vivo transduction experiments more feasible.

Animals↗
PubMed1997

Selective packaging of cargo molecules into endoplasmic reticulum-derived COPII vesicles.

Coated vesicles transport proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The formation of transport vesicles in vitro requires the incubation of an ER-membrane fraction with three protein fractions collectively known as coat protein II (COPII; Sar1p, Sec23p/Sec24p, and Sec13p/Sec31p). We used this assay to investigate how targeting [v-SNARE, vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor], putative adapter (e.g., Emp24p), and cargo molecules are captured into ER-derived COPII vesicles. Analysis of fusion proteins strongly suggests that the cytoplasmic domain of the v-SNARE protein Sec22p is required for its packaging into ER-derived COPII vesicles. We examined the packaging requirements for various molecules by individually titrating each of the COPII components. More Sar1p (the GTP-binding protein that initiates vesicles budding) is needed to package the membrane-associated v-SNAREs and Emp24p than is needed to package the soluble secretory protein glycosylated pro-alpha-factor (gp alphaF). Microsomes prepared from a strain overproducing Sec12p (the nucleotide exchange protein that recruits Sar1p to the ER) produce vesicles containing gp alphaF without the addition of exogenous Sar1p, whereas the v-SNAREs and Emp24p are not efficiently packaged under these conditions. Addition of Sar1p to these microsomes leads to increased packaging of v-SNAREs and Emp24p with no increase in the packaging of gp alphaF. Finally, we show that membranes prepared from strains with mutations in the SEC16 gene are more defective for the packaging of v-SNARE molecules and Emp24p than they are for the packaging of gp alphaF. These results point to the possibility that diverse signals or adapters participate in the capture of secretory and membrane cargo molecules into COPII transport vesicles.

Biological Transport↗
PubMed2003

High cloning capacity of in vitro packaged SV40 vectors with no SV40 virus sequences.

In vitro packaging of plasmid DNA using recombinant SV40 capsid proteins is a potentially useful procedure that overcomes some restrictions of the other SV40 systems such as the requirement for SV40 sequences and the limitation in size of DNA that can be packaged. The in vitro packaging system uses the four SV40 proteins (VP1, VP2, VP3, and agno) or VP1 only. The ability to confer drug resistance by three ABC transporter genes (MDR 1, MRP 1, or MXR) was determined using the surrogate fluorescent substrates rhodamine-123 or calcein AM and their specific inhibitors, or by using specific antibodies to the transporters to detect cell surface expression by fluorescence-activated cell sorter analysis (FACS). A green fluorescent protein plasmid (EGFP-C1) was also used to monitor gene transfer. The packaged plasmids ranged in size from 4.2 to 17.6 kb, and only slightly affected particle size as determined by electron microscopy. When 9.5 kb and larger plasmids were packaged using all SV40 proteins, MDR1 expression was decreased compared to VP1 alone. The size of the 15.2 kb DNA after packaging was the same as the original DNA. Packaging with SV40 capsid proteins in vitro does not require any SV40 sequences. Using either the MDR1 or the GFP gene we could demonstrate enhanced expression when cells were pretreated with phorbol 12-myristate 13-acetate (PMA) at low concentrations. Interferon-gamma did not alter expression. We conclude that in vitro packaging is more flexible then previously realized, permitting packaging of at least 17 kb plasmid DNA without the requirement for any viral sequences. This system combines efficient gene delivery of the SV40 viral vector with the presumed safety of nonviral vectors.

Cell Line↗
PubMed1994

Controlled trial evaluation of a General Practitioner education package on incontinence: use of a mailed questionnaire.

OBJECTIVE: To evaluate whether an education package will significantly improve General Practitioners' knowledge of the aetiology, diagnosis and treatment of incontinence. SUBJECTS AND METHODS: Of the 510 General Practitioners contacted 124 returned a 27 item questionnaire (q1). The doctors were randomized into a test group (n = 80) who received an education package and a control group (n = 44) who did not. Follow-up questionnaires (q2) were sent to those in the test group at 6 weeks and to those in the control group at 4 weeks post-randomization. The answers to 18 of the 27 questions were found in the education package and the remaining nine were not. The test group was asked to rate the usefulness of the package. RESULTS: There was a mean increase in scores for the two questionnaires of 5.2 in the test group compared with 0.4 in the control group. The average increase in scores in the test group was highly significant compared with that in the control group (P < 0.001). The results remained significant when the non-responders were included. The change in scores in the test group was significant only for those 18 questions with answers contained in the education package. There was no significant improvement between the two groups for the change in scores for the nine questions not answered in the education package (P = 0.74). General Practitioners gave the package a mean rating of 4.2/5 and it was still being used more than 6 weeks after delivery by at least 31 of 49 General Practitioners. CONCLUSIONS: For the General Practitioners who expressed an interest in receiving an education package on incontinence, and who answered the questionnaires, there was an improvement in the knowledge of incontinence directly attributable to the package.

Attitude of Health Personnel↗
PubMed2006

Translation of the flavivirus kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging.

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Animals↗
PubMed1992

Identification and characterization of a coronavirus packaging signal.

Previously, a mouse hepatitis virus (MHV) genomic sequence necessary for defective interfering (DI) RNA packaging into MHV particles (packaging signal) was mapped to within a region of 1,480 nucleotides in the MHV polymerase gene by comparison of two DI RNAs. One of these, DIssF, is 3.6 kb in size and exhibits efficient packaging, whereas the other, DIssE, which is 2.3 kb, does not. For more precise mapping, a series of mutant DIssF RNAs with deletions within this 1,480-nucleotide region were constructed. After transfection of in vitro-synthesized mutant DI RNA in MHV-infected cells, the virus product was passaged several times. The efficiency of DI RNA packaging into MHV virions was then estimated by viral homologous interference activity and by analysis of intracellular virus-specific RNAs and virion RNA. The results indicated that an area of 190 nucleotides was necessary for packaging. A computer-generated secondary structural analysis of the A59 and JHM strains of MHV demonstrated that within this 190-nucleotide region a stable stem-loop of 69 nucleotides was common between the two viruses. A DIssE-derived DI DNA which had these 69 nucleotides inserted into the DIssE sequence demonstrated efficient DI RNA packaging. Site-directed mutagenic analysis showed that of these 69 nucleotides, the minimum sequence of the packaging signal was 61 nucleotides and that destruction of the secondary structure abolished packaging ability. These studies demonstrated that an MHV packaging signal was present within the 61 nucleotides, which are located on MHV genomic RNA 1,381 to 1,441 nucleotides upstream of the 3' end of gene 1.

Animals↗
PubMed1994

Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants.

In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called psi that is required for packaging the viral RNA genome. Since the pol and env genes are dispensable for formation of RNA-containing particles, the gag gene product must have an RNA binding domain(s) capable of recognizing psi. To gain information about which portion(s) of Gag is required for RNA packaging in the avian sarcoma and leukemia virus system, we utilized a series of gag deletion mutants that retain the ability to assemble virus-like particles. COS cells were cotransfected with these mutant DNAs plus a tester DNA containing psi, and incorporation of RNA into particles were measured by RNase protection. The efficiency of packaging was determined by normalization of the amount of psi+ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of psi+ and psi- RNA in particles and in cells. The results indicate that much of the MA domain, much of the p10 domain, half of the CA domain, and the entire PR domain of Gag are unnecessary for efficient packaging. In addition, none of these deleted regions is needed for specific selection of the psi RNA. Deletions within the NC domain, as expected, reduce or eliminate both the efficiency and the specificity of packaging. Among mutants that retain the ability to package, a deletion within the CA domain (which includes the major homology region) is the least efficient. We also examined particles of the well-known packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather is the result of the cumulative effect of many point mutations throughout the gag gene.

Avian Sarcoma Viruses↗
PubMed1999

The nucleocapsid domain is responsible for the ability of spleen necrosis virus (SNV) Gag polyprotein to package both SNV and murine leukemia virus RNA.

Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.

Amino Acid Sequence↗