Packaging Defects Research Articles

Mutational Analyses of Packaging Signals in Influenza Virus PA, PB1, and PB2 Genomic RNA Segments

The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3' and 5' termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5' sites in the PA or PB1 coding regions but extended across the entire (80-base) 5' coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.

In vivo particle polymorphism results from deletion of a N-terminal peptide molecular switch in brome mosaic virus capsid protein

The interaction between brome mosaic virus (BMV) coat protein (CP) and viral RNA is a carefully orchestrated process resulting in the formation of homogeneous population of infectious virions with T = 3 symmetry. Expression in vivo of either wild type or mutant BMV CP through homologous replication never results in the assembly of aberrant particles. In this study, we report that deletion of amino acid residues 41–47 from the N-proximal region of BMV CP resulted in the assembly of polymorphic virions in vivo. Purified virions from symptomatic leaves remain non-infectious and Northern blot analysis of virion RNA displayed packaging defects. Biochemical characterization of variant CP by circular dichroism and MALDI-TOF, respectively, revealed that the engineered deletion affected the protein structure and capsid dynamics. Most significantly, CP subunits dissociated from polymorphic virions are incompetent for in vitro reassembly. Based on these observations, we propose a chaperon-mediated mechanism for the assembly of variant CP in vivo and also hypothesize that 41KAIKAIA 47 N-proximal peptide functions as a molecular switch in regulating T = 3 virion symmetry.

Molecular dynamics in semifluorinated‐side‐chain polysulfone studied by broadband dielectric spectroscopy

AbstractThe molecular dynamics of a set of polysulfone polymers have been studied with broadband dielectric spectroscopy. The materials comprise main‐chain polysulfones attached to semifluorinated (SF) oxydecylperfluorodecyl side chains. Through the analysis of the dielectric data together with information from differential scanning calorimetry and small‐angle X‐ray scattering, it can be shown that the oxydecylperfluorodecyl side chains are microphase‐separated and form nanodomains with an independent dynamic glass‐transition (δSF) relaxation. In addition to the α process (which is the glass transition of the main chain), other relaxation processes can be detected with Arrhenius‐type activation energies of 38–90 ± 2 kJ/mol. A γSF process, observed only in polysulfone with SF side chains, can be assigned to librational fluctuations of the perpendicular component of the fluoroalkyl group. A γB relaxation reflects fluctuations of the dipole moments (OSO and CO) of the polymer, whereas a β process, with high activation energies (>70 ± 2 kJ/mol), is assigned to packaging defects in the material. A detailed description of the processes is provided together with a proposed relaxation scheme. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 105: 201–207, 2007

Rab27b regulates number and secretion of platelet dense granules

The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27a(ash/ash) Rab27b(-/-)) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet alpha-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for delta-storage pool deficiency in humans.

Molecular mechanisms of simian immunodeficiency virus SIV agm RNA encapsidation

Primate lentiviruses are composed of several distinct lineages, including human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus SIVagm. HIV-1 and HIV-2 have significant differences in the mechanisms of viral RNA encapsidation. Therefore, the RNA packaging mechanisms of SIVagm cannot be predicted from the studies of HIV-1 and HIV-2. We examined the roles of the nucleocapsid (NC) zinc finger motifs on RNA packaging by mutating the conserved zinc finger (CCHC) motifs, and whether SIVagm has a preference to package RNA in cis by comparing the RNA packaging efficiencies of gag mutants in the presence of a wild-type vector. Our results indicate that the SIVagm NC domain plays an important role in Gag–RNA recognition; furthermore SIVagm is distinct from the other currently known primate lentiviruses as destroying either zinc finger motif in the NC causes very drastic RNA packaging defects. Additionally, trans-packaging is a major mechanism for SIVagm RNA encapsidation.

Portal fusion protein constraints on function in DNA packaging of bacteriophage T4

Architecturally conserved viral portal dodecamers are central to capsid assembly and DNA packaging. To examine bacteriophage T4 portal functions, we constructed, expressed and assembled portal gene 20 fusion proteins. C-terminally fused (gp20-GFP, gp20-HOC) and N-terminally fused (GFP-gp20 and HOC-gp20) portal fusion proteins assembled in vivo into active phage. Phage assembled C-terminal fusion proteins were inaccessible to trypsin whereas assembled N-terminal fusions were accessible to trypsin, consistent with locations inside and outside the capsid respectively. Both N- and C-terminal fusions required coassembly into portals with approximately 50% wild-type (WT) or near WT-sized 20am truncated portal proteins to yield active phage. Trypsin digestion of HOC-gp20 portal fusion phage showed comparable protection of the HOC and gp20 portions of the proteolysed HOC-gp20 fusion, suggesting both proteins occupy protected capsid positions, at both the portal and the proximal HOC capsid-binding sites. The external portal location of the HOC portion of the HOC-gp20 fusion phage was confirmed by anti-HOC immuno-gold labelling studies that showed a gold 'necklace' around the phage capsid portal. Analysis of HOC-gp20-containing proheads showed increased HOC protein protection from trypsin degradation only after prohead expansion, indicating incorporation of HOC-gp20 portal fusion protein to protective proximal HOC-binding sites following this maturation. These proheads also showed no DNA packaging defect in vitro as compared with WT. Retention of function of phage and prohead portals with bulky internal (C-terminal) and external (N-terminal) fusion protein extensions, particularly of apparently capsid tethered portals, challenges the portal rotation requirement of some hypothetical DNA packaging mechanisms.

Packaging Defect Prompts Recall of Procedure Trays

Packaging Defect Prompts Recall of Procedure Trays

Isolating failing sites in IC packages using time domain reflectometry: Case studies

This paper deals with Time Domain Reflectometry (TDR) technique with the aim to confirm that this tool should not be kept away from a non-destructive failure analysis process flow. An improvement of known comparative TDR methodology, the sequential comparative method, is introduced and several case studies illustrate its better efficiency to isolate complex packaging defects. Also, main limitations of the technique are studied and several hardware improvements are proposed, especially in terms of spatial resolution.

Non‐contact infrared detection of defects in electronic packaging

A non‐contact infrared detection by a steady direct current electro‐thermal technique is established to detect possible defects in electronic packaging. In the infrared detection process, researchers generally use the temperature rise attributed to the direct current source to estimate defects roughly. To detect defects accurately, the interference to the temperature rise due to parameters aside from defects (such as current inputs, defect types) needs to be filtered out. The so‐called “temperature defect influence factor T” (i.e. the ratio of temperature rise of a defective structure detected from experiment to that of a perfect one from finite element analysis) together with a rigorous detection criterion are thus devised in this work to detect defects. By depicting the contours of the temperature defect influence factor, the shape, size, number and location of defects in electronic packaging can be clearly detected using the detection procedure developed. As a case study, the detection of defects in thin quad flat package (TQFP) is performed. The results demonstrate that the present technique is feasible and effective for defect detection in electronic packaging.

Involvement of a bovine viral diarrhea virus NS5B locus in virion assembly.

A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-to-proline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans-packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly.

Automated defect recognition of C-SAM images in IC packaging using Support Vector Machines

Ultrasonic C-mode scanning acoustic microscopy (C-SAM) is widely used in the semiconductor industry for reliability testing and product inspection due to its ability to nondestructively detect defects in IC packaging. However, image interpretation and defect identification depend largely on the experience of operators, and there is no defect recognition system; this is partly due to current recognition systems, which are based on computer vision algorithms and are not robust for C-SAM images. A new robust defect recognition system and its application to C-SAM images are described in this paper. The iconic domain of two-dimensional C-SAM grey-level image analysis based on the non-linear Mumford-Shah model is used, and defect recognition is achieved through the use of Support Vector Machines (SVMs). The system is verified through experiments on a sequence of C-SAM images corrupted by synthetically generated noise, bias and different shape. The remarkable defect recognition rates achieved indicate that Support Vector Machines (SVMs) are suitable for IC package defect identification.

Effect of Microorganism Characteristics on Leak Size Critical to Predicting Package Sterility

The effects of microorganism size and motility on the leak size critical to the sterility of a package, along with the imposed pressure required to initiate liquid flow for the critical leak size, were measured. Pseudomonas fragi Lacy-1052, Bacillus atrophaeus ATCC 49337, and Enterobacter aerogenes ATCC 29007 were employed to assess package sterility. One hundred twenty-six 7-mm-long microtubes with interior diameters of 5, 10, and 20μm were used to simulate package defects. Forty-two solid microtubes were used as controls. No significant differences were found between sizes or motility statuses of test organisms with respect to loss of sterility as a result of microbial ingress into test cells with microtube interior diameters of 5, 10, and 20μm (P > 0.05). Interactions between the initiation of liquid flow as a result of applied threshold pressures and sterility loss for test cells were significant (P < 0.05).

Point mutations in exon I of the herpes simplex virus putative terminase subunit, UL15, indicate that the most conserved residues are essential for cleavage and packaging.

The herpes simplex virus UL15 and UL28 genes are believed to encode two subunits of the terminase involved in cleavage and packaging of viral genomes. Analysis of the UL15 protein sequence and its herpesvirus homologues revealed the presence of 20 conserved regions. Twelve of the twenty regions conserved among herpesviruses are also conserved in terminases from DNA bacteriophage. Point mutations in UL15 were designed in four conserved regions: L120N (CR1), Q205E (CR2), Q251E (CR3), G263A (CR3), and Y285S (CR4). Transfection experiments indicated that each mutant gene could produce stable UL15 protein at wild-type levels; however, only one mutant (Q251E) was able to complement the UL15-null virus. Each mutation was introduced into the viral genome by marker transfer, and all mutants except Q251E were unable to form plaques on Vero cells. Furthermore, failure to form plaques on Vero cells correlated with a defect in cleavage and packaging. Immunofluorescence experiments indicated that in cells infected with all mutant viruses the UL15 protein could be detected and was found to localize to replication compartments. Although wild-type and mutant Q251E were able to produce A, B, and C capsids, the rest of the mutants were only able to produce B capsids, a finding consistent with their defects in cleavage and packaging. In addition, all mutant UL15 proteins retained their ability to interact with B capsids. Therefore, amino acid residues 120, 205, 263, and 285 are essential for the cleavage and packaging process rather than for association with capsids or localization to replication compartments.

Involvement of the matrix and nucleocapsid domains of the bovine leukemia virus Gag polyprotein precursor in viral RNA packaging.

The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging.

Importance of Vp1 calcium-binding residues in assembly, cell entry, and nuclear entry of simian virus 40.

For polyomaviruses, calcium ions are known to be essential for virion integrity and for the assembly of capsid structures. To define the role of calcium ions in the life cycle of the virus, we analyzed simian virus 40 (SV40) mutants in which structurally deduced calcium-binding amino acids of Vp1 were mutated singly and in combination. Our study provides evidence that calcium ions mediate not only virion assembly but also the initial infection processes of cell entry and nuclear entry. Mutations at Glu48, Glu157, Glu160, Glu216, and/or Glu330 are correlated with different extents of packaging defects. The low packaging ability of mutant E216R suggests the need to position the Glu216 side chain for proper virion formation. All other mutants selected for further analysis produced virus-like particles (VLPs) but were poorly infectious. The VLPs of mutant E330K could not attach to or enter the cell, and mutant E157A-E160A and E216K VLPs entered the cell but failed to enter the nucleus, apparently as a result of premature VLP dissociation. Our results show that five of the seven acidic side chains at the two calcium-binding sites-Glu48 and Glu330 (site 1), Glu157 and Glu160 (site 2), and Glu216 (both sites)-are important for SV40 infection. We propose that calcium coordination imparts not only stability but also structural flexibility to the virion, allowing the acquisition or loss of the ion at the two sites to control virion formation in the nucleus, as well as virion structural alterations at the cell surface and in the cytoplasm early during infection.

Application of Fluid Modeling To Determine Threshold Leak Size for Liquid Foods

In this study, the mechanism by which a package defect converts to a leaker was examined in an effort to develop a relationship between threshold leak size and loss of package sterility. The threshold leak size is the hole size at which the onset of leakage occurs. The threshold pressure is the pressure required to initiate a leak. Leak initiation was studied in terms of the interaction between three components: liquid attributes of liquid food products, defect size, and pressures required to initiate liquid flow. Liquid surface tension, viscosity, and density values were obtained for 16 liquids. The imposed pressures required to initiate flow through microtubes with interior diameters of 0, 2, 5, 7, 10, 20, and 50μm were measured with the use of 63 test cells filled with safranin red dye, tryptic soy broth, and distilled water with surface tensions of 18.69, 44.09, and 64.67 mN/m, respectively. Significant differences (P < 0.05) between threshold pressures observed for safranin red dye, tryptic soy broth, and distilled water were found. Liquids with low surface tensions, such as safranin red dye, required significantly lower threshold imposed pressures than did liquids with high surface tensions, such as distilled water (P < 0.05). An equation to quantify the relationship between liquid surface tension, threshold imposed pressure, and defect size was developed. Threshold pressures observed were not significantly different (P > 0.05) from those predicted by the equation. Imposed pressures and vacuums generated within packages during random vibration and sweep resonance tests were measured for brick-style aseptic packages (250 ml), metal cans (76.2 by 114.3mm [425 ml]), 1-qt gable-top packages (946 ml), 0.5-gal gable-top packages (1.89 liters), and 1-gal milk jugs (4.25 liters). Significant differences between packages were found with respect to observed generated pressures during vibration testing (P < 0.05). An equation to calculate threshold size on the basis of liquid surface tension and imposed pressure was established.

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding.

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.

Analysis of RNA Packaging in Wild-Type and Mosaic Protein Capsids of Flock House Virus Using Recombinant Baculovirus Vectors

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two positive-sense RNA molecules, RNA1 (replicase gene) and RNA2 (coat protein gene), which are encapsidated into a single virion. Expression of coat protein in Sf21 cells using a baculovirus vector results in formation of virus-like particles (VLPs) whose capsids are structurally indistinguishable from native virions. However, RNA packaging is not specific for RNA2, the coat protein message. Using ribonuclease protection assays, we showed that the fraction of RNA2 in VLPs is 19% relative to the amount present in a population of native virions. To investigate possible reasons for the reduced level of RNA2, we generated two new baculovirus vectors, AcR1delta and AcR2delta, expressing the replicase gene and the coat protein gene, respectively. The inserted genes carried the self-cleaving hepatitis delta ribozyme sequence at the 3' end to allow for synthesis of RNA1 and RNA2 transcripts with authentic 3' ends. Infection of Sf21 cells with AcR2delta yielded VLPs that contained 66% RNA2 relative to native virions. Coinfection of Sf21 cells with AcR1delta and AcR2delta launched self-directed FHV replication and resulted in formation of particles most of which contained RNA1 and RNA2. However, a small fraction of particles containing cellular RNA was detected as well. The latter particles could be eliminated by infecting Sf21 cells with AcR1delta followed by transfection with in vitro synthesized transcripts of RNA2. We have further utilized this system to show that two coat protein deletion mutants with distinct RNA packaging defects form mosaic virus capsids but do not complement each other to rescue specific packaging of FHV RNAs.

Finding internal component flaws

A packaging defect inside a component used in an automotive application often has no immediate electrical consequences. One example is a delamination in a plastic IC. If the delamination is between the plastic moulding compound and the face of the silicon chip, the IC may test normal electrically.

Three-dimensional x-ray laminography as a tool for detection and characterization of BGA package defects

Non-destructive examination of the layers of a built-up substrate was achieved using three-dimensional (3-D) x-ray microlaminography, with successful separation of layers as thin as 8 /spl mu/m. The same technology was used to create reconstructed images of both surface and internal details of inner solder balls in a ball grid array package soldered to a printed circuit board. Microlaminography was also used to identify bond-wire shorts in the plane of the solder resist of a ball grid array assembly, and these were subsequently verified by destructive physical analysis. This plane is 20 /spl mu/m thick and immediately adjoining a plane of copper traces; the success demonstrates the capability of microlaminography to resolve and separate very fine detail internally within IC structures. The limits of capability of this machine were also determined; it was found that a crack of approximately 5 /spl mu/m wide in a copper trace of a BGA was not detected by the machine. As an introduction, the technology and methodology of 3-D x-ray microlaminography are explained. The results from a microlaminography system adapted for failure analysis in integrated circuit packaging are presented. It is shown that such results could not be extracted by two-dimensional (2-D) x-ray or other nondestructive methods.