[Standardization of Biobank and Potential of IVD Industries to Utilize Biospecimens].

Many biobanks that have stored human clinical biospecimens have been established in Japan since 2005. Those biospecimens were mainly used in academia to perform basic research. The use of those biospeci- mens by industries, especially pharmaceutical or in vitro diagnostic companies, was restricted for ethical rea- sons. In this review, we discuss the importance of the standardization of the quality of biospecimens and workflow at biobanks for the utilization of specimens, and the possibility and issues regarding specimen use by industries.

Effects of cinacalcet on bone mineral density and bone markers in hemodialysis patients with secondary hyperparathyroidism.

BACKGROUND: Cinacalcet markedly reduces the serum intact parathyroid hormone (PTH) level of hemodialysis (HD) patients with secondary hyperparathyroidism. Parathyroidectomy also reduces the serum intact PTH level of HD patients and it increases their bone mineral density (BMD). However, there is little information about the effect of cinacalcet on BMD or on the associations between bone markers and BMD in HD patients. METHODS: We performed a 1-year cohort study of 25 HD patients who had a serum intact PTH level above 300 pg/ml during treatment by conventional therapies, such as with active vitamin D, and cinacalcet was prescribed for 14 of them. BMD of the femoral neck and the serum levels of two circulating bone markers, alkaline phosphatase (ALP) and bone-specific alkaline phosphatase (BSAP), were measured before and after treatment. The other 11 HD patients without cinacalcet treatment were defined as control group. RESULTS: BMD significantly increased by 7.3 % during the 1 year of treatment in the cinacalcet group and decreased by 6.2 % during the same period in the control group, and cinacalcet therapy was independently associated with the changes in BMD after multiple regression analysis that included intact PTH (ß = 7.57, P < 0.01). In the cinacalcet group, the serum ALP levels (R(2) = 0.315, P < 0.05) and BSAP levels (R(2) = 0.682, P < 0.01) levels were significantly negatively correlated with the changes in BMD, but the serum intact PTH levels were not significantly associated with the changes in BMD (R(2) = 0.011, P = 0.72). CONCLUSIONS: One year of treatment with cinacalcet increased the BMD of the femoral neck in the HD cohort, especially in the patients who had higher serum ALP and BSAP levels at baseline.

Inhibitory effects of the transcription factor Ets-1 on the expression of type I collagen in TGF-ß1-stimulated renal epithelial cells.

Extracellular matrix (ECM) production and epithelial-mesenchymal transition (EMT) are important for phenotypic conversion in normal development and disease states such as tissue fibrosis. Transforming growth factor-ß1 (TGFß1) is one of the most potent inducers of ECM proteins, and its role in the pathogenesis of fibrosis is well established. Ets family is involved in a diverse array of biologic functions including cellular growth, migration, and differentiation. In the present study, we investigated whether Ets-1 has a role in ECM production and EMT in human renal tubuloepithelial cells (HKC cells). TGFß1 treatment increases Ets-1 expression and nuclear translocation in the HKC cells. Overexpression of recombinant Ets-1 suppressed transcription of α2(I) collagen (COL1A2) and type I collagen production in the TGFß1-activated HKC cells. From the experiments using specific inhibitors against Smad3 or mitogen-activated protein (MAP) kinase pathways, Ets-1 has an inhibitory role for COL1A2 transcription and the p38 MAPK pathway participates in the negative contribution of Ets-1 in TGFß1/Smad3-activated renal cells.

Suppression of renal fibrosis by galectin-1 in high glucose-treated renal epithelial cells.

Diabetic nephropathy is the most common cause of chronic kidney disease. We investigated the ability of intracellular galectin-1 (Gal-1), a prototype of endogenous lectin, to prevent renal fibrosis by regulating cell signaling under a high glucose (HG) condition. We demonstrated that overexpression of Gal-1 reduces type I collagen (COL1) expression and transcription in human renal epithelial cells under HG conditions and transforming growth factor-ß1 (TGF-ß1) stimulation. Matrix metalloproteinase 1 (MMP1) is stimulated by Gal-1. HG conditions and TGF-ß1 treatment augment expression and nuclear translocation of Gal-1. In contrast, targeted inhibition of Gal-1 expression reduces COL1 expression and increases MMP1 expression. The Smad3 signaling pathway is inhibited, whereas two mitogen-activated protein kinase (MAPK) pathways, p38 and extracellular signal-regulated kinase (ERK), are activated by Gal-1, indicating that Gal-1 regulates these signaling pathways in COL1 production. Using specific inhibitors of Smad3, ERK, and p38 MAPK, we showed that ERK MAPK activated by Gal-1 plays an inhibitory role in COL1 transcription and that activation of the p38 MAPK pathway by Gal-1 plays a negative role in MMP1 production. Taken together, two MAPK pathways are stimulated by increasing levels of Gal-1 in the HG condition, leading to suppression of COL1 expression and increase of MMP1 expression.

Correlation of new bone metabolic markers with conventional biomarkers in hemodialysis patients.

BACKGROUND: New bone metabolic markers have become available clinically for evaluating chronic kidney disease mineral and bone disorders (CKD-MBD). The aim of this study was to correlate these new bone metabolic markers with conventional markers in regular hemodialysis (HD) patients. METHODS: One hundred forty three HD patients underwent cross-sectional assessment. Two bone formation markers, bone-specific alkaline phosphatase (BAP) and osteocalcin (OC), and one bone resorption marker, amino-terminal telopeptides of type 1 collagen (NTx), were selected for study. RESULTS: Both circulating OC and NTx levels showed positive correlations with serum intact parathyroid hormone (iPTH) levels. The levels of NTx and OC showed a strongly positive correlation, although they are known to be markers of different aspects of bone metabolism: bone formation and resorption. Patients with high iPTH (≥300pg/mL) had significantly higher levels of all the three bone markers compared with patients with low or normal iPTH . CONCLUSION: Serum OC and NTx levels may be useful markers of serum iPTH levels for evaluating bone turnover in HD patients and may eventually prove useful in the management of patients with CKD-MBD.

A single amino acid alteration in cytoplasmic domain determines IL-2 promoter activation by ligation of CD28 but not inducible costimulator (ICOS).

The CD28 family molecules, CD28, and inducible costimulator (ICOS) all provide positive costimulatory signals. However, unlike CD28, ICOS does not costimulate IL-2 secretion. The YMNM motif that exists in the CD28 cytoplasmic domain is a known binding site for phosphatidylinositol 3-kinase (PI3-K) and Grb2. ICOS possesses the YMFM motif in the corresponding region of CD28 that binds PI3-K but not Grb2. We postulated that the reason that ICOS does not have the ability to induce IL-2 production is because it fails to recruit Grb2. To verify this hypothesis, we generated a mutant ICOS gene that contains the CD28 YMNM motif and measured IL-2 promoter activation after ICOS ligation. The results indicated that ICOS became competent to activate the IL-2 promoter by this single alteration. Further analysis demonstrated that Grb2 binding to ICOS was sufficient to activate the NFAT/AP-1 site in the IL-2 promoter and that the cytoplasmic domain of CD28 outside of the YMNM motif is required for activation of the CD28RE/AP-1 and NF-kappaB sites. Together, these observations lead us to believe that the difference of a single amino acid, which affects Grb2 binding ability, may define a functional difference between the CD28- and ICOS-mediated costimulatory signals.

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA.

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

Direct measurement of reactive oxygen species in leukocytes during hemodialysis therapy.

OBJECTIVES: Both chronic kidney disease (CKD) and hemodialysis (HD) are reported to elevate oxidative stress. Available evidence for oxidative stress is indirect measurement of oxidative stress as accumulation of byproducts by reactive oxygen species (ROS). We aimed to examine the effect of CKD and HD on ROS levels in circulating leukocytes and to compare those with conventional oxidative stress marker, F2-isoprostane, in HD patients. METHODS: Using flowcytometry techniques, ROS levels in circulating leukocytes can be directly measured in 16 HD patients and 12 healthy volunteers. We also measured circulating F2-isoprostanes levels in both groups. RESULTS: HD patients demonstrated a significant increase in serum levels of F2-isoprostanes. The direct measurement of ROS levels in leukocytes showed increase in HD patients compared to the control; 1.91-fold in polymorphonuclear leukocytes (PMN), 1.06-fold in lymphocytes, and 1.35-fold in monocytes. Significant difference between the two groups could be observed only in PMN. The ROS levels in all three fractions of leukocytes showed negative correlations with serum F2-isoprostane levels but the ROS levels only in PMN showed significant correlation (r(2) = 0.774, P = 0.001). CONCLUSIONS: Our results indicate that direct measurement of the ROS levels in circulating leukocytes by flowcytometry is a useful method to examine oxidative stress during HD procedure. The ROS levels in circulating leukocytes showed negative correlation with serum F2-isoprostane levels.

Eicosapentaenoic Acid (EPA) Decreases the All-Cause Mortality in Hemodialysis Patients.

OBJECTIVE: Atherosclerosis, which causes cardiovascular disease, is a major cause of death in hemodialysis (HD) patients. Eicosapentaenoic acid (EPA), an anti-hyperlipidemic agent, is known to have antioxidative or anti-inflammatory effects, resulting in improvements in atherosclerosis. In the present study, we examined whether EPA improves the all-cause mortality in patients receiving regular HD therapy. METHODS: We enrolled 176 patients treated with maintenance HD therapy and performed a longitudinal observational cohort study for three years. We divided the patients into two groups based on whether or not the received EPA treatment [EPA(+) and EPA(-), respectively]. The primary end-point was all-cause death. We also matched the two groups using propensity score matching and examined the effect of EPA. RESULTS: Before matching, the all-cause mortality rates were 24.0% in the EPA(+) and 11.8% in the EPA(-) groups, which were significantly different (p=0.044). After propensity score matching, the EPA(+) group still showed a significantly better prognosis than the EPA(-) group (p=0.038). A multivariate analysis showed that EPA treatment significantly reduced the risk of all-cause mortality both before and after propensity score matching. CONCLUSION: EPA treatment is independently associated with lower mortality in HD patients.

Change in the expressed gene patterns of the wing disc during the metamorphosis of Bombyx mori.

The present study was conducted to clarify what occurs during the metamorphosis of the imaginal disc in insects. To understand the metamorphosis on a molecular level, the changes in expression profiles in the imaginal disc during metamorphosis were investigated. For this purpose, we constructed cDNA libraries from four different stages of wing discs of Bombyx mori, sequenced about 1000 cDNAs randomly collected from each library, and constructed a database of expressed sequence tags (EST). The morphological changes and expression profiles from EST were compared during those four stages. Microarray analysis was applied to quantify the expression of each gene in each stage in order to confirm whether the expression of the genes identified from EST was induced by 20-hydroxyecdysone (20E) in a stage-specific manner. Wing discs showed dynamic morphogenesis in 4-5 days during the preparatory stage of metamorphosis, which was under the control of an ecdysteroid. Different expressed profiles were observed in each of the four different stages by comparison of each EST clone. These profiles reflected the morphological changes of the Bombyx wing disc during metamorphosis. The results of expression profiles from the four stages suggested that the V4 stage was cell proliferating; W0, proliferating and the beginning of differentiation; W2, morphologically changing; W3, cuticle secreting. Microarray analysis showed the effectiveness of its application on 20E induction of genes in wing discs. The wing disc of B. mori is an exceptionally suitable system for understanding the relationship between morphological changes and the distribution of mRNA.

A Bombyx mori gene, BmChi-h, encodes a protein homologous to bacterial and baculovirus chitinases.

We have cloned and characterized a novel chitinase gene (BmChi-h) from the silkworm, Bombyx mori. BmChi-h cDNA has an open reading frame of 1,665 nucleotides, encoding a protein of 555 amino acid residues. The predicted protein shared extensive similarities with bacterial and baculovirus chitinases in both amino acid sequences (73% identity with Serratia marcescens chiA and 63% with Autographa californica nucleopolyhedrovirus chiA) and domain architectures. BmChi-h was a single-copy gene and located on chromosome 7. The expression of BmChi-h mRNA was observed in a stage- and tissue-specific manner that was almost identical to that of another chitinase gene previously cloned from B. mori. We further determined the overall genomic organization of BmChi-h. There was no intron in the ORF of BmChi-h. However, BmChi-h was transcribed from three promoters, which generated three isoforms in the 5'-UTR of the transcript. Phylogenetic analysis suggested that ancestral species of B. mori acquired the chitinase gene from a bacterium or an ancestral baculovirus via horizontal gene transfer.

Serum osteocalcin levels are useful as a predictor of cardiovascular events in maintenance hemodialysis patients.

BACKGROUNDS: Osteocalcin (OC) is a known bone metabolic marker and a regulator of glucose and fat metabolisms. Although bone and energy metabolisms are known risk factors for cardiovascular disease (CVD) in hemodialysis (HD) patients, few studies have examined the correlation between OC and CVD. The purpose of this study was to investigate the impact of serum OC levels on the emergence of new CVD events in HD patients. METHODS: We designed a longitudinal, observational cohort study in which the study patients were divided into low- and high-serum OC groups based on a median serum OC level of 71.5 ng/ml. RESULTS: Cardiovascular disease events were observed in 29 of 126 patients (23.0 %). The number of cumulative CVD events in the low-serum OC group was significantly higher than that in the high-serum OC group, as evaluated by the Kaplan-Meier method (p = 0.0021, log-rank test). Multivariate Cox proportional hazards analysis demonstrated that a low level of serum OC is a significant predictor of a higher incidence of CVD events [hazard ratio, 2.925; 95 % confidence interval, 1.048-9.066; p = 0.0401] after adjustment. CONCLUSION: Serum OC level is a significant, independent prognostic factor for CVD events in maintenance HD patients. OC may be useful in predicting new CVD events in HD patients.

Study of discrepancies between recorded and actual blood flow in hemodialysis patients.

Adequate blood flow (Qb) is necessary for effective hemodialysis (HD). Aim of the study was to examine relationship between the actually delivered Qb (dQb) and reported Qb (rQb) with dialysis machine. One hundred HD patients with arteriovenous fistula were enrolled. Delivered Qb was measured at the beginning and end of each HD session. dQb/rQb < 1 indicated a discrepancy between actual dQb and rQb reported using a dialysis machine. In addition, dQb/rQb was examined in HD patients using needles of different gauges during treatment. The average levels of dQb/rQb at start and end of HD session were 1.01 ± 0.04 and 0.98 ± 0.05, respectively. In the 16 gauge and 17 gauge needle groups, the percentage of patients with dQb/rQb < 1 increased in accordance with the increase in rQb or as the HD session progressed. In the 15 gauge needle group, the percentage of patients with dQb/rQb < 1 was <50% at any level of rQb. Selection of needle gauge is important factors for determining actual dQb in HD patients.

Flowcytometric analysis of lymphocytapheresis in a patient with recurrent FSGS after renal transplant.

Frequently, focal segmental glomerulosclerosis (FSGS) recurs after renal transplantation, resulting in poor graft survival. Pathological mechanisms of the recurrence are still unknown, but both B and T cell disorders are suspected based on much evidence. This supports theoretical benefits using plasma exchange (PE) and lymphocytapheresis (LCAP). A renal transplant was performed for a 35-year-old woman, who suffered steroid-resistant FSGS and developed to chronic kidney disease stage 5D at 31 years old. We treated the patient with recurrent FSGS by LACP and examined whether peripheral neutrophils were dynamically changed after the therapy. Further, we performed flowcytometric analysis to examine lymphocyte fractions before and after LCAP. The decrease of helper (CD4 positive) and memory (CD4 and CD45RO positive) T cells was prominent after LCAP. Although B cells were at the nadir because of rituximab treatment, LCAP also decreased peripheral B cells. These suggest that LCAP has the potential to suppress the activities of recurrent FSGS after renal transplant.

Cardiovascular autonomic neuropathy studied by a laser-Doppler blood flowmeter in hemodialysis patients.

OBJECTIVE: Orthostatic hypotension during a hemodialysis (HD) session affects not only the modality but daily quality of life for HD patients because many of them have combined dysfunction of both sympathetic and parasympathetic nervous systems. Although various non-invasive methods have been applied for the evaluation of autonomic function, no monitor has been devised for measuring the dysfunction during blood purification therapy. PATIENTS AND METHODS: We evaluated the usefulness of laser-Doppler blood flowmeter (LDF) for measuring autonomic function of stable 34 regular HD patients and 24 healthy controls. The LDF device was applied for autonomic test by measuring periflux blood flow decreasing velocity (PDV) accompanied with Valsalva maneuver. We also evaluated the correlation between PDV and conventional tests for atherosclerosis. RESULTS: The average PDV (3.79±1.77) in HD population level was significantly lower than that of healthy controls (8.72±6.00). We also found a significant correlation between PDV and conventional methods such as heart rate variability and ankle-brachial blood pressure index. CONCLUSION: Measurement of PDV by LDF is as useful as a conventional method for evaluating autonomic function in HD patients. The convenience of the device offers the benefit of daily and frequent measurement of autonomic dysfunction.

Analysis of plasma adipocytokines related to clinical and laboratory data in the maintenance hemodialysis patients.

BACKGROUND: Atherosclerotic vascular diseases such as cerebrovascular and cardiovascular diseases are major causes for fatality of hemodialysis (HD) patients. Since adipocytokines are key players for arteriosclerosis in the concept of metabolic syndrome (MetS), we aimed to determine whether circulating levels of major three adipocytokines, adiponectin, TNF-alpha, and leptin, could be associated with various parameters and clinical events in HD patients who are diagnosed as MetS using a new criteria designed for the Japanese population. PATIENTS AND METHODS: We enrolled 53 very stable patients under maintenance HD at Minami-Senju Hospital. Basically, clinical and laboratory data were taken just before HD therapy. HD sessions were performed regularly and all the participants took oral administration and injection as usual. A cross-sectional study was performed to evaluate clinical and laboratory data related to three major adipocytokines, adiponectin, TNF-alpha and leptin. RESULTS: We observed no significant differences of three adipocytokines when the participants were divided in accordance with existence of MetS or past cerebrocascular/cardiovascular diseases. Only the serum adiponectin levels were significantly different in two groups categorized by existence of diabetes mellitus. Serum triglycerides (TG) were significantly correlated with two circulating adipocytokines, adiponectin (r=-0.328, p<0.016) and leptin (r=0.397, p<0.003), when we analyzed all 53 patients together. CONCLUSION: Plasma adiponectin and leptin are expected as contributors related to dyslipidemia, suggesting these may be targets of prevention of vascular diseases in maintenance HD patients.

A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids.

DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3.

The single-stranded DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus consists of 385 amino acid residues, forms oligomers, and promotes Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region (residues 28 to 326) that is relatively resistant to proteolysis. In contrast, the N-terminus (27 residues) and C-terminal portion (59 residues) are not involved in interaction with DNA and are readily accessible to proteolytic digestion. Circular dichroism analyses showed that LEF-3 is a folded protein with an estimated alpha-helix content of more than 40%, but it is structurally unstable and undergoes unfolding in aqueous solutions at temperatures near 50 degrees C. Unfolding eliminated the LEF-3 domains that are resistant to proteolysis and randomized the digestion pattern by trypsin. The structural transition was irreversible and was accompanied by the generation of high molecular weight (MW) complexes. The thermal treatment inhibited DNA-binding and unwinding activity of LEF-3 but markedly stimulated its annealing activity. We propose that the shift in LEF-3 activities resulted from the generation of the high MW protein complexes, that specifically stimulate the annealing of complementary DNA strands by providing multiple DNA-binding sites and bringing into close proximity the interacting strands. The unfolded LEF-3 was active in a strand exchange reaction suggesting that it could be involved in the production of recombination intermediates.

RACK1 binds to Smad3 to modulate transforming growth factor-beta1-stimulated alpha2(I) collagen transcription in renal tubular epithelial cells.

Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter.

Characterization of the role of very late expression factor 1 in baculovirus capsid structure and DNA processing.

Very late expression factor 1 (VLF-1) of Autographa californica multiple nucleopolyhedrovirus is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production. In this report, we show that a vlf-1 knockout bacmid was able to synthesize viral DNA at levels similar to that detected for a gp64 knockout bacmid that served as a noninfectious control virus. Additionally, analysis of replicated bacmid DNA by field-inversion gel electrophoresis indicated that VLF-1 is not required for synthesizing high-molecular-weight intermediates that could be resolved into unit-length genomes when cut at a unique restriction site. However, immunoelectron microscopic analysis revealed that in cells transfected with a vlf-1 knockout bacmid, aberrant tubular structures containing the capsid protein vp39 were observed, suggesting that this virus construct was defective in producing mature capsids. In contrast, rescuing the vlf-1 knockout bacmid construct with a copy of VLF-1 that carries a mutation of a highly conserved tyrosine (Y355F) was sufficient to restore the production of nucleocapsids with a normal appearance, but not infectious virus production. Furthermore, the results of a DNase I protection assay indicated that the DNA packaging efficiency of the VLF-1(Y355F) virus construct was similar to that of the gp64 knockout control. Finally, a recombinant virus containing a functional hemagglutinin epitope-tagged version of VLF-1 was constructed to investigate the association of VLF-1 with the nucleocapsid. Analysis by immunoelectron microscopy of Sf-9 cells infected with this virus showed that VLF-1 localized to an end region of the nucleocapsid. Collectively, these results indicate that VLF-1 is required for normal capsid assembly and serves an essential function during the final stages of the DNA packaging process.