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PubMed1999

An intact TAR element and cytoplasmic localization are necessary for efficient packaging of human immunodeficiency virus type 1 genomic RNA.

Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5' splice site, others have suggested that sequences upstream of the splice site may also play an important role. In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged. Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem. Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.

5' Untranslated Regions↗

PubMed2002

cis-Acting elements important for retroviral RNA packaging specificity.

Spleen necrosis virus (SNV) proteins can package RNA from distantly related murine leukemia virus (MLV), whereas MLV proteins cannot package SNV RNA efficiently. We used this nonreciprocal recognition to investigate regions of packaging signals that influence viral RNA encapsidation specificity. Although the MLV and SNV packaging signals (Psi and E, respectively) do not contain significant sequence homology, they both contain a pair of hairpins. This hairpin pair was previously proposed to be the core element in MLV Psi. In the present study, MLV-based vectors were generated to contain chimeric SNV/MLV packaging signals in which the hairpins were replaced with the heterologous counterpart. The interactions between these chimeras and MLV or SNV proteins were examined by virus replication and RNA analyses. SNV proteins recognized all of the chimeras, indicating that these chimeras were functional. We found that replacing the hairpin pair did not drastically alter the ability of MLV proteins to package these chimeras. These results indicate that, despite the important role of the hairpin pair in RNA packaging, it is not the major motif responsible for the ability of MLV proteins to discriminate between the MLV and SNV packaging signals. To determine the role of sequences flanking the hairpins in RNA packaging specificity, vectors with swapped flanking regions were generated and evaluated. SNV proteins packaged all of these chimeras efficiently. In contrast, MLV proteins strongly favored chimeras with the MLV 5'-flanking regions. These data indicated that MLV Gag recognizes multiple elements in the viral packaging signal, including the hairpin structure and flanking regions.

3T3 Cells↗

PubMed2003

Minimal cis-acting elements required for adenovirus genome packaging.

The design of drugs for treatment of virus infections and the exploitation of viruses as drugs for treatment of diseases could be made more successful by understanding the molecular mechanisms of virus-specific events. The process of assembly, and more specifically packaging of the genome into a capsid, is an obligatory step leading to future infections. To enhance our understanding of the molecular mechanism of packaging, it is necessary to characterize the viral components necessary for the event. In the case of adenovirus, sequences between nucleotides 200 and 400 at the left end of the genome are essential for packaging. This region contains a series of redundant bipartite sequences, termed A repeats, that function in packaging. Synthetic packaging sequences made of multimers of a single A repeat substitute for the authentic adenovirus packaging domain. A repeats are binding sites for the CCAAT displacement protein and the viral protein IVa2. Several lines of evidence implicate these proteins in the packaging process. It was not known, however, whether other cis-acting elements play a role in the packaging process as well. We utilized an in vivo approach to address the role of the inverted terminal repeats and the covalently linked terminal proteins in packaging of the adenovirus genome. Our results show that these elements are not necessary for efficient packaging of the viral genome. A significant implication of these results applicable to gene therapy vector design is that the linkage of the adenovirus packaging domain to heterologous DNA sequences should suffice for targeting to the viral capsid.

Adenoviruses, Human↗

PubMed2004

Identification of the 15FRFG domain in HIV-1 Gag p6 essential for Vpr packaging into the virion.

The auxiliary regulatory protein Vpr of HIV-1 is packaged in the virion through interaction with the Gag C-terminal p6 domain. Virion packaging of Vpr is critical for Vpr to exert functions in the HIV-1 life cycle. Previous studies suggest that Vpr interacts with a (Lxx)4 domain in p6 for virion packaging. In the present study, mutational analysis of HIV-1 Gag p6 domain was performed in the context of the HIV-1 genome to examine the effect on virion packaging of Vpr. Surprisingly, Ala substitutions for Leu44 and Phe45 in the (Lxx)4 domain or deletion of the whole (Lxx)4 domain (amino acid #35-52 of the Gag p6 domain) did not affect Vpr virion packaging. Vpr virion packaging was normal when amino acid #1-23 of the Gag p6 domain was preserved. Most importantly, Ala substitutions for Phe15, Arg16 and Phe17 in the context of amino acid #1-23 of the Gag p6 domain abolished Vpr virion packaging. Single Ala substitutions for Phe15 and Phe17 also abolished Vpr virion packaging, whereas Ala substitution for Arg16 had no effect. Our studies have revealed a novel signal sequence for Vpr packaging into the HIV-1 virion. The 15FRFG domain in p6 resembles the FxFG repeat sequences commonly found in proteins of the nuclear pore complex. These results have provided novel insights into the process of virion packaging of Vpr and suggest for the first time that Vpr may recognize the FxFG domain for both virion packaging and association with nuclear pores.

Alanine↗

PubMed2002

Food and drink packaging: who is complaining and who should be complaining.

This paper reports a questionnaire survey of shoppers at four supermarket stores in the UK. Regression analyses were used to determine factors in consumer complaints with the packaging of food and drink products. Neuroticism and treatment in hospital for an accident involving packaging in the last 3 years were significant associates. Analyses on who was most likely to suffer accidents or injuries while opening food and drink packaging indicated that handedness (that is, being left handed made accidents more likely) and scoring low on a measure of personal control during decision-making were significant associates. Analyses on who suffered the most serious accidents and injuries whilst opening food and drink packaging revealed that the decision-making style of social resistance (that is, the extent to which you resist asking for help from others) was related to susceptibility to the most severe accidents. These findings imply that individual factors (including both personal characteristics and personality traits) should be taken into account when considering the openability of packaging. The results of this study indicate that manufacturers should ensure that novel packaging closures have been tested on left handers in order to discover any unique difficulties for this sub-group, that packaging opening tools specifically for left-handed people should be made available and that manufacturers of packaging should always look at a 'worst case scenario' for their packaging closures as it is evident that some people will continue to struggle with difficult packaging until either they open it or they have injured themselves.

Accidents↗

PubMed1997

Migration and sorption phenomena in packaged foods.

Rapidly developing analytical capabilities and continuously evolving stringent regulations have made food/package interactions a subject of intense research. This article focuses on: (1) the migration of package components such as oligomers and monomers, processing aids, additives, and residual reactants in to packaged foods, and (2) sorption of food components such as flavors, lipids, and moisture into packages. Principles of diffusion and thermodynamics are utilized to describe the mathematics of migration and sorption. Mathematical models are developed from first principles, and their applicability is illustrated using numerical simulations and published data. Simulations indicate that available models are system (polymer-penetrant) specific. Furthermore, some models best describe the early stages of migration/sorption, whereas others should be used for the late stages of these phenomena. Migration- and/or sorption-related problems with respect to glass, metal, paper-based and polymeric packaging materials are discussed, and their importance is illustrated using published examples. The effects of migrating and absorbed components on food safety, quality, and the environment are presented for various foods and packaging materials. The impact of currently popular packaging techniques such as microwavable, ovenable, and retortable packaging on migration and sorption are discussed with examples. Analytical techniques for investigating migration and sorption phenomena in food packaging are critically reviewed, with special emphasis on the use and characteristics of food-simulating liquids (FSLs). Finally, domestic and international regulations concerning migration in packaged foods, and their impact on food packaging is briefly presented.

Environmental Exposure↗

PubMed2004

Biopolymer-based antimicrobial packaging: a review.

The term antimicrobialpackaging encompasses any packaging technique(s) used to control microbial growth in a food product. These include packaging materials and edible films and coatings that contain antimicrobial agents and also techniques that modify the atmosphere within the package. In recent years, antimicrobial packaging has attracted much attention from the food industry because of the increase in consumer demand for minimally processed, preservative-free products. Reflecting this demand, the preservative agents must be applied to packaging in such away that only low levels of preservatives come into contact with the food. The film or coating technique is considered to be more effective, although more complicated to apply. New antimicrobial packaging materials are continually being developed. Many of them exploit natural agents to control common food-borne microorganisms. Current trends suggest that, in due course, packaging will generally incorporate antimicrobial agents, and the sealing systems will continue to improve. The focus of packaging in the past has been on the appearance, size, and integrity of the package. A greater emphasis on safety features associated with the addition of antimicrobial agents is perhaps the next area for development in packaging technology.

Anti-Bacterial Agents↗

PubMed1985

Tamper-resistant packaging: is it elder-resistant, too?

A tamper-resistant package has an indicator or barrier to entry that, if breached or missing, can reasonably be expected to provide visible evidence to consumers that tampering has occurred. Regulations to implement tamper-resistant packaging on all over-the-counter drugs and certain cosmetics began in February 1983. Tamper-resistant packaging, like child-resistant packaging, may impede access by the elderly and other adults who have mental, motor, and/or sensory disabilities. This article describes: the reasons for the increasing use of over-the-counter medications in the prevention and treatment of the major causes of morbidity and mortality in the elderly; the types of difficulties encountered in opening tamper-resistant packaging; the causes of these inaccessibility problems; the methods to correct them; and testing procedures to determine whether the existing designs of tamper-resistant packaging are accessible by the elderly and adults with selected disabilities. Testing of individual types of tamper-resistant packaging, multiple types of tamper-resistant packagings on the same container, and combinations of tamper-resistant packagings and child-resistant packagings on groups of normal elderly people and those with selected disabilities is necessary to guarantee accessibility to the growing number of therapeutically efficacious nonprescription medications used by these populations.

Aged↗

PubMed2000

The effect of PVC packaging on the acidity of 0.9% saline.

Intravenous fluids in polyvinyl chloride (PVC) packaging are known to be acidic. We proposed to determine the effect of PVC packaging on the pH of 0.9% saline solutions by comparing the predicted and measured pH of 0.9% saline equilibrated with atmospheric carbon dioxide and the measured pH of commercial solutions of 0.9% saline in PVC and polypropylene packaging. Calculation of pH was made from available physical chemistry constants and data. Measurement was made of the pH of 12 samples of prepared 0.9% saline equilibrated with atmospheric carbon dioxide. Comparison with the pH of seven commercial samples of saline in PVC packaging for intravenous use was undertaken. Further comparison was made between commercial samples of 0.9% saline in PVC or polypropylene packaging. The calculated pH of 0.9% saline was 5.61 at 20 degrees C. The median pH of the prepared samples was statistically significantly less acidic than the median pH of the PVC packaged samples for intravenous use: 5.47 vs 4.60, P < 0.05. The median pH of the PVC packaged saline was also statistically significantly more acidic than the pH of the polypropylene packaged saline: 4.62 vs 5.71, P < 0.05. The acidity of the intravenous solutions of 0.9% saline packaged in PVC was much greater than expected and is only partially explained by dissolved carbon dioxide. This acidity could be a result of packaging in PVC.

Acids↗

PubMed2006

Use of pre-packaged chloroquine for the home management of presumed malaria in Malagasy children.

OBJECTIVE: The main objective of this study was to assess the quality of home malaria management with pre-packaged chloroquine in two areas in the Moramanga district of Madagascar. The knowledge, attitude and practices of care providers in terms of home treatment options were evaluated and compared. The availability of treatment options by studying retailers and community-based service providers was also investigated. METHODS: A cross-sectional investigation in two communities, in the hamlets and villages located close to carers, retailers, community-based service providers and primary health centres was carried out. RESULTS: Carers in the two districts were equally well aware of the use of pre-packaged chloroquine. Their first response to the onset of fever was to treat children with this antimalarial drug at home. The dose administered and treatment compliance were entirely satisfactory (100%) with pre-packaged chloroquine and rarely satisfactory (1.6% to 4.5%) with non pre-packaged chloroquine. In cases of treatment failure, the carers took patients to health centres. Chloroquine was supplied principally by private pharmacies and travelling salesmen selling unpackaged chloroquine tablets. Non pre-packaged chloroquine was the most common drug used at health centres. The frequency of positive rapid malaria tests (P = 0.01) was significantly higher in children treated with non pre-packaged chloroquine (38%) than in children treated with pre-packaged chloroquine (1.3%). CONCLUSION: Home malaria management should be improved in Madagascar. Efforts should focus on communication, the training of community-based service providers, access to pre-packaged drugs and the gradual withdrawal of pre-packaged chloroquine and its replacement by pre-packaged artemisinin-based combination therapies.

Adult↗

PubMed2002

Mechanisms underlying sawtoothed grain beetle (Oryzaephilus surinamensis [L.]) (Coleoptera: Silvanidae) infestation of consumer food packaging materials.

The sawtoothed grain beetle, Oryzaephilus surinamensis (L.), is an extremely destructive pest of packaged consumer food products. The beetle is not believed to chew directly through packaging materials, but to use openings or flaws in damaged or improperly sealed packages to gain entry. We investigated the behavioral mechanisms by which the sawtoothed grain beetle infests packages with flaws. Significantly more sawtoothed grain beetles infested consumer food packages that had been punctured with 0.4 mm diameter holes, to simulate packaging flaws that preclude adults, than when packages had no flaws. In a test arena, females laid more eggs into or near the hole in a plastic packaging film, when they were able to contact the food through the hole than when they could not contact the food. First instar larvae placed either 1 mm or 1 cm away entered holes when food was present, indicating that packages could become infested if eggs were laid near holes. In the absence of food, neither adults nor larvae responded to holes. This study has shown the importance of sound packaging in preventing insect infestation.

Animals↗

PubMed2003

Microbiological and physicochemical properties of fresh retail cuts of beef packaged under an advanced vacuum skin system and stored at 4 degrees C.

The effect of an advanced vacuum skin packaging system on the microbiological and physicochemical properties of fresh retail cuts of beef (including meat portions from six different anatomical regions) stored at 4 degrees C was compared with the effect of traditional vacuum packaging. The vacuum skin packaging system whose effect on meat quality was evaluated in this work displayed two remarkable features: (i) the instantaneous heating of the lower surface of the upper film of the package before the film descended over the meat surface and (ii) the tight disposition of the plastic film on the meat surface, which avoided wrinkles and purges. Throughout storage at 4 degrees C, rates of bacterial growth were statistically significantly slower in beef portions processed with the vacuum skin packaging system than in those processed with traditional vacuum packaging, with average differences of 2.07, 1.60, and 1.25 log CFU/g in counts of aerobic mesophiles, anaerobes, and lactic acid bacteria, respectively. pH values were statistically significantly lower for beef portions packaged with the vacuum skin system than for those that were vacuum packaged in the traditional manner, probably because of the relative predominance of lactic acid bacteria observed in such samples, which coincided with both higher meat firmness values and a slower meat tenderization process. The vacuum skin system prevented the appearance of undesirable coloration on the meat surface and also significantly improved the commercial color of the meat as determined on the basis of luminosity (L*) and the redness (a*). Overall, the quality (as determined by microbiological and physicochemical analyses and by visual examination) of fresh retail cuts of beef packaged with the vacuum skin system and stored at 4 degrees C was higher than that of meat samples processed with the traditional vacuum-packaging system.

Animals↗

PubMed2006

Fate of inoculated Escherichia coli O157:H7, cultured under different conditions, on fresh and decontaminated beef transitioned from vacuum to aerobic packaging.

This study evaluated the behavior of Escherichia coli O157:H7 during aerobic storage, after storage in vacuum packages, on beef inoculated with cultures prepared (35 degrees C, 24 h) in tryptic soy broth without dextrose (TSB), nonacid hot water carcass decontamination runoff fluids (washings; pH 6.0; WASH), cells from biofilms formed on stainless steel coupons in WASH (WETB), or WETB dried (25 degrees C, 12 h) before harvesting of cells (DRYB). These inocula were applied to fresh beef pieces (40 cm2), which were then left untreated or treated by immersion in hot water (75 degrees C) followed by 2% lactic acid (55 degrees C; hot water/lactic acid [HW/LA]), for 30 s each. Inoculated samples were vacuum packaged and stored at 0 (30, 60, or 90 days), 4 (7 or 14 days), or 12 degrees C (4 or 8 days) and subsequently transferred to retail packages for aerobic storage at 7 degrees C for 5 days. Populations of E. coli O157:H117, regardless of inoculum type, remained generally unchanged (P > 0.05) after aerobic storage (7 degrees C, 5 days) of untreated or HW/LA-treated beef samples previously stored in vacuum packages at 0 or 4 degrees C. However, reductions in E. coli O157:H7 levels were generally obtained when vacuum packaged, untreated beef samples previously stored at 12 degrees C were transitioned to aerobic conditions. Additionally, despite similar (P > 0.05) levels of E. coli O157:H7 cells of TSB, WASH, WETB, and DRYB origin on vacuum-packaged, untreated samples after 8 days of storage at 12 degrees C, subsequent aerobic storage resulted in larger (P < 0.05) reductions of cells of WETB and DRYB origin than for cells of TSB and WASH origin. For HW/LA-treated beef previously stored at 12 degrees C in vacuum packages, populations of E. coli O157:H7 remained largely unchanged after aerobic storage in retail packages. Results thus indicated that aerobic storage of beef (7 degees C, 5 days) previously stored in vacuum packages at 0 or 4 degrees C did not lead to E. coli O157:H7 population changes, whereas transition from vacuum packages stored under mildly abusive temperature (12 degrees C) to aerobic storage may have caused injury and death to the pathogen.

Animals↗

PubMedDate not supplied

Development and characterization of a minimal inducible packaging cell line for simian immunodeficiency virus-based lentiviral vectors.

BACKGROUND: Lentiviral vectors allow gene transfer into non-dividing cells. Further development of these vector systems requires stable packaging cell lines that enable adequate safety testing. METHODS: To generate a packaging cell line for vectors based on simian immunodeficiency virus (SIV), expression plasmids were constructed that contain the codon-optimized gag-pol gene of SIV and the gene for the G protein of vesicular stomatitis virus (VSV-G) under the control of an ponasterone-inducible promoter. Stable cell lines expressing these packaging constructs were established and characterized. RESULTS: The RT activity and vector titers of cell clones stably transfected with the inducible gag-pol expession plasmid could be induced by ponasterone by more than a factor of 1000. One of these clones was subsequently transfected with the ponasterone-inducible VSV-G expression plasmid to generate packaging cells. Clones of the packaging cells were screened for vector production by infection with an SIV vector and subsequent induction by ponasterone. In the supernatant of selected ponasterone-induced producer clones vector titers of more than 1x10(5) transducing units/ml were obtained. Producer cell clones were stable for at least five months, as tested by vector production. CONCLUSIONS: The packaging cells described should be suitable for most preclinical applications of SIV-based vectors. By avoiding regions of high homology between the vector and the packaging constructs, the design of the SIV packaging cell line should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the SIV vector constructs that can be packaged.

Cell Line↗

PubMed1995

Structural and functional domains of the large subunit of the bacteriophage T3 DNA packaging enzyme: importance of the C-terminal region in prohead binding.

During head assembly of phage T3, DNA is packaged into a preformed protein shell, called the prohead, with the aid of non-capsid packaging proteins, the products of genes 18 and 19 (gp18 and gp19). We have developed a defined system, composed of purified gp18,gp19 and proheads for in vitro packaging of T3 DNA. Our previous results using the defined in vitro system indicate the sequential events in DNA packaging: the packaging proteins, gp18 and gp19, bind DNA and proheads, respectively. These complexes associate to form a direct precursor complexes for DNA translocation into the head. The formation of the precursor complexes requires ATP as an allosteric effector. Subsequent DNA translocation is driven by ATP hydrolysis. gp19 is an ATP binding protein that plays multiple roles in DNA packaging through interaction with ATP. gp19 changes its conformation by binding to ATP, as judged from the analysis of limited proteolysis. Sites cleaved by limited proteolysis were determined and mapped on the gp19 polypeptide (586 amino acid residues) to image the conformational change of gp19 induced by ATP. C-Terminal fragments generated by trypsin digestion bound the prohead and inhibited DNA packaging by intact gp19 in a competitive manner. On the other hand, N-terminal fragments did not bind the prohead nor did they inhibit DNA packaging. These results define a prohead binding domain at the C terminus of gp19. To identify the prohead binding domain more precisely, deletion mutants lacking the last 10 and 15 amino acids (gp19-delta C10 and gp19-delta C15, respectively) of the extreme C terminus of gp19 were constructed. Limited tryptic digestion patterns of these mutant proteins in the presence or absence of ATP were basically the same as those of gp19-wt, indicating that the conformation and its ATP response were not changed by these deletions. gp19-delta C15 lacked prohead binding activity and, therefore, DNA packaging activity. gp19-delta C10 had significant DNA packaging activity although it was reduced to one-tenth of that of gp19-wt. These results indicate that a C-terminal region of residues L571 to D576 of gp19 is crucial for prohead binding and that the last ten residues D577 to W586 of the C terminus seems to be important in stable binding of gp19 to the prohead.

Adenosine Triphosphate↗

PubMed1994

Hydrogen exchange dynamics of the P22 virion determined by time-resolved Raman spectroscopy. Effects of chromosome packaging on the kinetics of nucleotide exchanges.

We describe the application of laser Raman spectroscopy to probe hydrogen isotope exchange dynamics of nucleic acid and protein constituents in a double-stranded DNA virus, the icosahedral bacteriophage P22. The Raman dynamic method employs a dialysis flow cell to control D2O efflux into an H2O solution of the virus sample while the rates of deuterium exchange of protons in the viral nucleic acid and protein molecules are measured spectrophotometrically in real time. The method provides structural and kinetic information about three different and distinct classes of exchangeable protons of the native virion: (1) labile imino (NH) and amino (NH2) protons of the bases which participate in Watson-Crick hydrogen bonding in the packaged genome; (2) pseudolabile purinic (8CH) protons that line the major groove of packaged P22 DNA; and (3) main-chain amide (NH) protons of viral subunits comprising the shell that encapsidates the DNA. The results obtained on P22 demonstrate that interchange of aqueous solvent with the virion interior is rapid and complete. We find that while labile protons of packaged DNA exchange rapidly, most amide protons in capsid subunits are resistant to solvent-catalyzed exchange. Further, stereospecific retardation of exchange is observed for major-groove protons of the packaged P22 genome. The quantitative measurements can be summarized and interpreted as follows. (1) Imino and amino protons of all bases in packaged P22 DNA exchange more rapidly (approximately 2-fold faster) than the corresponding protons in unpackaged P22 DNA. Remarkably, packaging actually accelerates labile imino and amino hydrogen exchanges of the viral DNA, an effect which can be attributed to selective stabilization in the packaged chromosome of a base-pair open state (breathing model). (2) Conversely, purine 8CH exchange rates in packaged P22 DNA are significantly retarded in comparison to those of unpackaged P22 DNA. The observed 8CH exchange retardation effects are similar for both adenine and guanine residues, indicating that they do not originate from purine-specific interactions but probably reflect steric shielding of the major groove of packaged DNA from free access to solvent. This effect is likely distributed throughout the 43,400 base-pair genome. (3) Only a small population (approximately 15 to 20%) of subunit amide protons exchanges within the time frame of complete exchange of all protons of packaged P22 DNA. Complete exchange of the capsid is not achieved even after several months of incubation at 40 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage P22↗

PubMed1998

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range </=10 to 200 molecules of SN packaged per head, and independent of proteolytic cleavage of SN from the IPIII portion of the fusion, favoring a discontinuous packaged DNA structure. Rods of B-form DNA could be envisioned as protected from digestion, whereas bent or kinked DNA would be more susceptible to the diffusible SN. Such discontinuous packaged DNA structures are favored for phage T4 by a number of lines of evidence.

Bacteriophage T4↗

PubMed2002

Human immunodeficiency virus type 1 preferentially encapsidates genomic RNAs that encode Pr55(Gag): functional linkage between translation and RNA packaging.

Full-length retroviral RNA serves as both messenger and genomic RNA. Therefore, an unspliced RNA could play both roles: viral mRNA could be bound in cis by the same Gag polyprotein that it produced, becoming a packaged genomic RNA. To test this possibility, we used in vivo packaging experiments which coexpressed wild-type NL4-3 RNA and NL4-3-based mutant RNA that, ideally, could not translate Gag. However, mutating the gag initiator produced a mutant (pNLX) that expressed a truncated Gag, Gag*, initiated at methionine 10 in the CA region (142 of Pr55(Gag)). Gag* can be rescued into virions by Gag and, as it contains the NC domain, could package RNA in cis. To eliminate NC and the CA dimerization domain, a nonsense mutation in CA at residue 99 was introduced into pNLX to produce pNLXX, which expresses an RNA that should only be packaged in trans. Cotransfection packaging experiments revealed that wild-type genomic RNA was packaged at an 8-fold greater level than NLXX RNA given equal expression of both RNAs. Experiments that varied the relative amounts of these RNAs in the cell found that the wild-type RNA was encapsidated with a packaging preference (i.e., the relative amount of this RNA in virions versus cells) of 6- to 13-fold over the NLXX RNA, showing that the NLXX RNA did not efficiently compete with NL4-3 RNA. These data suggest that the wild-type RNA's ability to express Pr55(Gag) and, by inference, actively translate Gag confers an advantage in packaging over the nearly identical NLXX RNA. In contrast, the NLX RNA competed with wild-type RNA at a 1-to-3 preference. This ratio is similar to the amounts of Gag* rescued by Gag, suggesting that the presence of Gag* assists in the encapsidation of NLX RNA. Together, our data link translation and particle formation to the packaging of viral RNA and support a model of cis packaging where nascent Gag proteins encapsidate their cognate RNA.

Cell Line↗