Search

Tacrolimus (TAC) is essential for prophylaxis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic cell transplantation (allo-HSCT). We have sometimes observed large fluctuations in TAC concentration. However, links between the variability in the concentration or the concentration/dose (C/D) ratio of TAC and clinical complications remain ambiguous. To clarify relationships between various parameters of TAC and early complications such as pre-engraftment immune reactions/engraftment syndrome, aGVHD, and transplant-associated thrombotic microangiopathy (TA-TMA), a total of 146 patients who underwent allo-HSCT were included. Intrapatient variabilities in the concentration and C/D ratio of TAC were estimated by intrapatient mean absolute deviation (iMAD). The mean concentration and C/D ratio of TAC were not significantly different between with and without complications. A strong association was observed between greater iMAD for TAC C/D ratio from days 15 to 21 and the development of TA-TMA. iMAD values for TAC C/D ratio of 11.4 or greater was a risk factor for TA-TMA and the cumulative incidence of nonrelapse mortality (NRM) was significantly higher in patients with iMAD values for TAC C/D ratio of 11.4 or greater. Intrapatient variability in the C/D ratio of TAC was associated with the incidence of TA-TMA and NRM and might be useful for predicting TA-TMA.

One major cause of treatment-related death is transplant-associated thrombotic microangiopathy (TA-TMA). Because of difficulties with diagnosis, many criteria for TA-TMA have been defined. Some patients clinically suspected as TA-TMA have been treated as TA-TMA regardless of TA-TMA criteria fulfillment (clinical-TMA). To examine sensitivities of TA-TMA criteria for clinical-TMA, we retrospectively evaluated 160 patients undergoing allogeneic hematopoietic stem cell transplantation by five major TA-TMA criteria and compared them with clinical-TMA. Cumulative incidences of TA-TMA and non-relapse mortality (NRM) were widely diverse between criteria. Thirty-eight patients fulfilled one or more TA-TMA criteria (total-TMA), and 12 of them fulfilled only one criterion. In patients with total-TMA, thrombocythemia, serum creatinine > 1.5 × baseline, and proteinuria were especially repeatedly observed among TA-TMA criteria. Ninety-two percent of clinical-TMA patients were classified as patients with total-TMA, and high NRM incidences were exhibited in patients with total-TMA even without clinical-TMA. Hematopoietic cell transplant-comorbidity index ≥ 3, nutritional risk index

Permanent impairment (PI) of vital organs is one of the transplantation-related health problems affecting the quality of life and morbidity even in patients who do not develop graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT), but no data are available on PI of multiple organs. This retrospective study aimed to estimate a novel composite endpoint of PI-free, relapse-free survival (PIRFS) in 164 allo-HCT recipients. We defined PI as26% to 30% impairment of the whole person in 6 vital organs using the whole person impairment rating. Conventional GVHD-free/relapse-free survival (GRFS) and PIRFS at 5 years were 33.8% (95% confidence interval [CI], 26.5% to 41.3%) and 40.6% (95% CI, 32.6% to 48.4%), respectively. In the whole cohort, PIRFS was higher than GRFS at any time after allo-HCT. However, PIRFS was lower than GRFS after day 397 post-transplantation in patients who underwent umbilical cord blood transplantation (UCBT). In UCBT recipients, 5-year GRFS and PIRFS were 47.6% (95% CI, 34.3% to 59.7%) and 39.2% (95% CI, 26.6% to 51.5%), respectively. The cumulative incidence of PI after 5 years was 20.9% (95% CI, 13.7% to 29.0%) in patients surviving for ≥6 months without relapse. The multivariate analysis revealed that high disease risk (hazard ratio [HR], 1.91; 95% CI, 1.26 to 2.88; P.01) and Karnofsky Performance Status score ≤90% at transplantation (HR, 1.73; 95% CI, 1.14 to 2.63; P = .01) were correlated with the lower PIRFS, whereas UCBT (HR, 2.35; 95% CI, 1.11 to 4.99; P = .03), grade III-IV acute GVHD by day 180 (HR, 3.59; 95% CI, 1.04 to 12.4; P = .04), and thrombotic microangiopathy by day 180 (HR, 2.74; 95% CI, 1.10 to 6.87; P = .03) were significantly correlated with a higher incidence of PI. More than 20% of long-term survivors had PI. Our data suggest that PIRFS is a useful endpoint for assessing long-term transplantation success from a different perspective than has been established previously.

Morphogenesis and organ development should be understood based on a thorough description of cellular dynamics. Recent studies have explored the dynamic behaviors of mammalian neural progenitor cells (NPCs) using slice cultures in which three‐dimensional systems conserve in vivo‐like environments to a considerable degree. However, live observation of NPCs existing truly in vivo, as has long been performed for zebrafish NPCs, has yet to be established in mammals. Here, we performed intravital two‐photon microscopic observation of NPCs in the developing cerebral cortex of H2B‐EGFP or Fucci transgenic mice in utero. Fetuses in the uterine sac were immobilized using several devices and were observed through a window made in the uterine wall and the amniotic membrane while monitoring blood circulation. Clear visibility was obtained to the level of 300 μm from the scalp surface of the fetus, which enabled us to quantitatively assess NPC behaviors, such as division and interkinetic nuclear migration, within a neuroepithelial structure called the ventricular zone at embryonic day (E) 13 and E14. In fetuses undergoing healthy monitoring in utero for 60 min, the frequency of mitoses observed at the apical surface was similar to those observed in slice cultures and in freshly fixed in vivo specimens. Although the rate and duration of successful in utero observations are still limited (33% for ≥10 min and 14% for 60 min), further improvements based on this study will facilitate future understanding of how organogenetic cellular behaviors occur or are pathologically influenced by the systemic maternal condition and/or maternal‐fetal relationships., Intravital two‐photon microscopic observation of division of neural progenitor cells in the developing cerebral cortex was performed in utero.

Nutritional status is an important component of cancer care, and malnutrition itself can cause death in 10% to 20% of cancer patients. A nutritional risk index (NRI) is a useful tool for nutritional assessment of cancer patients. This study aimed to evaluate the impact of pretransplant NRI values on outcomes of allogeneic hematopoietic cell transplantation (allo-HSCT). One hundred sixty patients who underwent allo-HSCT between January 2008 and July 2017 at Konan Kosei Hospital were included in this single-center, retrospective analysis. NRI was calculated at the beginning of the conditioning regimen. The patients were divided into high NRI (NRI ≥ 97.5) and low NRI (NRI97.5) groups, and overall survival (OS), nonrelapse mortality (NRM), and cumulative incidences of acute and chronic graft-versus-host disease (GVHD) were evaluated. Two-year OS rates were 76% (95% confidence interval [CI], 63% to 83%) and 50.4% (95% CI, 38% to 62%) in the high NRI and low NRI groups, respectively (P.001). One-year cumulative incidences of NRM were 7.9% (95% CI, 3.5% to 15%) and 23% (95% CI, 14% to 33%; P = .014) and 2-year cumulative relapse rates were 17% (95% CI, 10% to 26%) and 32% (95% CI, 21% to 43%; P = .10) in the high NRI and low NRI groups, respectively. The multivariate analysis indicated low NRI was a significant risk factor for OS and NRM. Conversely, high NRI was associated with increased incidences of grades II to IV acute GVHD and chronic GVHD. Additionally, the subgroup analysis according to stem cell source revealed a significant benefit of higher NRI on survival only in umbilical cord blood recipients. Overall, these results suggest that pretransplant NRI might predict OS and NRM after allo-HSCT.

Acute myeloid leukemia (AML) associated with double-minute chromosomes (dmin) is a rare condition and has a poor prognosis. A 68-year-old man with leukocytosis and thrombocytopenia was admitted to our hospital. Bone marrow aspiration showed that 79.5% of myeloblasts were positive for myeloperoxidase. The patient was diagnosed with acute myeloid leukemia (French-American-British classification: M2, World Health Organization classification: AML, not otherwise specified, AML with maturation). Chromosomal analysis revealed the presence of dmin: 45, X, -Y, 5-33 dmin. Fluorescence in situ hybridization revealed multiple MYC signals, and spectral karyotyping showed that dmin was derived from chromosome 8. These findings indicated resistance to chemotherapy alone. After the standard induction therapy with daunorubicin and cytarabine, the number of myeloblasts in the bone marrow decreased, and the amplified MYC signals disappeared. Then, the patient achieved complete remission. Reportedly, most patients with AML correlated with dmin have a complex karyotype, except for this case. Owing to the absence of a complex karyotype, the patient had good sensitivity to chemotherapy. Further studies with a larger population of patients with AML associated with dmin, but without complex karyotypes, should be conducted to accurately predict prognosis in such cases.

Blastic plasmacytoid dendritic cell neoplasm is a rare disease with difficulty in diagnosis, and clinical courses of patients with BPDCN vary widely. We report a case of a 63-year-old man with BPDCN showing severe tumor lysis syndrome (TLS). BPDCN with high tumor burden should be carefully considered to prevent TLS.

The combination of methotrexate (MTX) and a calcineurin inhibitor is widely used for GVHD prophylaxis in umbilical cord blood transplantation (UCBT). However, the optimal MTX dosage for GVHD prophylaxis in UCBT remains unclear. In the present study, we investigated the impact of MTX dosage on clinical outcomes following UCBT in a single-center retrospective study. Of 70 UCBT recipients included in this study, 37 received MTX at doses of 10 mg/m2 on day 1, and 7 mg/m2 on days 3 and 6 (low-dose MTX: LD-MTX), and 33 received MTX at doses of 15 mg/m2 on day 1, and 10 mg/m2 on days 3 and 6 (standard-dose MTX: SD-MTX), in addition to tacrolimus (TAC). Grade 3–4 acute GVHD and/or transplant-related complications with endothelial cell damage were considered severe transplant-related complications. Univariate analysis findings revealed that the risk of grade 3–4 acute GVHD was significantly lower in the SD-MTX group than in the LD-MTX group (P = 0.013). Multivariate analysis findings revealed that SD-MTX was significantly associated with a lower incidence of severe transplant-related complications (HR = 0.25; 95% CI, 0.07–0.87; P = 0.029). We conclude that SD-MTX in combination with tacrolimus is optimal for GVHD prophylaxis in UCBT.

Reduced pulmonary function is commonly observed after allogeneic hematopoietic stem cell transplantation (HSCT); however, its relationship with the development of noninfectious pulmonary complications (NIPCs) is unclear, and the impact of changes in pulmonary function test (PFT) values on HSCT outcome remains controversial. We conducted a retrospective study including 150 patients to investigate changes in PFTs and impact on clinical outcome. PFT data at around 1 year after HSCT were available in 84 patients, and showed a significant time-dependent decline in percentage predicted forced expiratory volume in 1 s and other parameters. We focused on %FEV1, calculated decline of %FEV1 from pretransplant to around 1 year after HSCT (Δ%FEV1), and divided patients into good-Δ%FEV1 or poor-Δ%FEV1 groups, using a cut-off point of 20% decline of Δ%FEV1. In the poor-Δ%FEV1 group, half of the patients developed NIPCs. In the good-Δ%FEV1 group, PFT values were maintained, whereas those of the poor-Δ%FEV1 group declined significantly. Multivariate analysis showed that busulfan use was a risk factor for %FEV1 decline, and poor-Δ%FEV1 was a risk factor for overall survival. These data indicate that decline of %FEV1 may be a useful indicator of pulmonary damage after HSCT, and is strongly associated with busulfan use.

The hematopoietic cell transplantation-specific comorbidity index (HCT-CI) has been recently proposed to predict the probability of nonrelapse mortality (NRM) and overall survival (OS) in allogeneic hematopoietic stem cell transplantation (HSCT). However, the usefulness of the HCT-CI in single-unit umbilical cord blood transplantation (UCBT) remains unclear. We investigated the impact of the HCT-CI on the clinical outcomes of allogeneic HSCT in a single-center retrospective study including 53 recipients of UCBT (UCBT group) and 90 recipients of other HSCT (non-UCBT group). In the non-UCBT group 2-year OS rates for HCT-CI score 3 and ≥3 were 67% (n = 74; 95% confidence interval [CI], 54% to 78%) and 26% (n = 16; 95% CI, 7% to 51%), respectively (P = .001). In the UCBT group these rates were 66% (n = 39; 95% CI, 48% to 79%) and 69% (n = 14; 95% CI, 36% to 87%), respectively (P = .73). In the non-UCBT group 1-year NRM rates for HCT-CI score 3 and ≥3 were 14% (95% CI, 6.4% to 22%) and 37% (95% CI, 14% to 61%), respectively (P = .02). In the UCBT group these rates were 6.1% (95% CI, 3.4% to 24%) and 7.7% (95% CI, .4% to 29%), respectively (P = .78). Using multivariate analysis we showed that HCT-CI score ≥ 3 was significantly associated with lower OS (hazard ratio, 3.06; 95% CI, 1.47 to 6.38; P = .003) and higher NRM (hazard ratio, 2.87; 95% CI, 1.18 to 6.96; P = .02) for the non-UCBT group. UCBT showed good OS with low incidence of NRM, even in patients with high HCT-CI scores. Altogether, we propose single-unit UCB to be a promising stem cell source for improving survival in patients with multiple comorbidities.

A 64-year-old man with acute myeloid leukemia underwent umbilical cord blood transplantation (UCBT). After 11 months of complete remission (CR) following UCBT, the bone marrow showed 7.5% myeloblasts. CR was obtained after a single course of azacitidine monotherapy, but the myeloblasts gradually increased in the blood. We made a diagnosis of acute megakaryoblastic leukemia derived from donor cell with a fluorescence in situ hybridization (FISH) analysis of the sex chromosomes and an immunophenotypic analysis. Azacitidine was administered again and produced a therapeutic effect of stable disease. This case suggests that azacitidine may be a useful therapy for patients with acute megakaryoblastic leukemia in situations in which intensive chemotherapy and transplantation are not indicated.

Cryptogenic organizing pneumonia (COP) after allogeneic hematopoietic stem cell transplantation (HSCT) is characterized by frequent recurrence. Few studies have examined onset and recurrence patterns of COP after HSCT. We investigated the clinical features of COP after HSCT in a single-center retrospective study including 165 consecutive patients who underwent allogeneic HSCT. Eighteen patients (11%) developed COP after HSCT. Hypoxemia and pleural effusion at the onset of COP were significantly associated with umbilical cord blood transplantation (P = 0.002 and P = 0.002, respectively). Recurrence of COP was observed in six patients and significantly associated with the presence of chronic graft-versus-host disease (cGVHD; P = 0.013) and stem cell sources other than umbilical cord blood (P = 0.038). Four patients with COP died of pulmonary failure after recurrence of COP. No patients who underwent umbilical cord blood transplantation experienced recurrence of COP. These findings suggest that the clinical features at the onset of COP may depend on stem cell sources. Moreover, both stem cell source and the absence or presence of cGVHD may affect COP recurrence, indicating the need to develop treatment strategies against COP according to stem cell source and risk of cGVHD.

Introduction The combination of methotrexate (MTX) and a calcineurin inhibitor is widely used for GVHD prophylaxis in umbilical cord blood transplantation (UCBT). However, the optimal MTX dosage for GVHD prophylaxis in UCBT remains unclear. Methods We investigated the impact of the MTX dosage on clinical outcomes of UCBT. We retrospectively analyzed a cohort of 70 patients who received first UCBT between October 2002 and December 2017 in our hospital. Of the 70 recipients, 37 received MTX at doses of 10 mg/m2 on day 1, and 7 mg/m2 on days 3 and 6 (low-dose MTX: LD-MTX), and 33 received MTX at doses of 15 mg/m2 on day 1, and 10 mg/m2 on days 3 and 6 (standard-dose MTX: SD-MTX), in combination with tacrolimus (TAC).The following conditions within day 180 were considered to be transplant-related complications with endothelial cell damage: sinusoidal occlusive syndrome, systemic transplant-associated microangiopathy (TAM), intestinal TAM, capillary leak syndrome, idiopathic pneumonia syndrome, and diffuse alveolar hemorrhage. We defined grade 3-4 acute GVHD and/or transplant-related complications with endothelial cell damage as severe transplant-related complications in this study. Results The characteristics of patients and transplantation procedures were comparable between the two groups. T-cell depletion was not used as GVHD prophylaxis in our study. The median follow-up period for surviving patients was 1504 days (range, 302-3864). The cumulative incidence of neutrophil engraftment by day 42 was 97.3% (95% CI, 71.5%-99.8%) for the LD-MTX group and 93.9% (95% CI, 73.1%-99.8%) for the SD-MTX group (P = 0.36). The cumulative incidence of platelet engraftment by day 60 was 78.4% (95% CI, 60.6%-88.8%) for the LD-MTX group and 87.9% (95% CI, 69.0%-95.6%) for the SD-MTX group (P = 0.40). Univariate analysis findings revealed that the risk of grade 3-4 acute GVHD was significantly lower in the SD-MTX group than in the LD-MTX group (P = 0.013). Multivariate analysis findings revealed that SD-MTX was significantly associated with a lower incidence of severe transplant-related complications (HR = 0.25; 95% CI, 0.07-0.87; P = 0.029). Non-relapse death was not observed in patients with standard-risk disease in the SD-MTX group (n = 22). There was no significant difference between the two groups in the 2-year incidence of relapse: 27.0% (95% CI, 13.9%-42.0%) for the LD-MTX group and 15.3% (95% CI, 5.4%-29.7%) for the SD-MTX group (P = 0.55). Conclusion Our study demonstrated that the GVHD prophylaxis consisting of SD-MTX and TAC led to a significantly lower incidence of severe acute GVHD and/or TRC-EC than the combination of LD-MTX and TAC without increasing the risk of graft failure and relapse. Thus, while our findings need to be further verified by multicenter studies with larger cohorts of patients, for now SD-MTX is optimal in combination with TAC for GVHD prophylaxis in UCBT.

To achieve highly sensitive and comprehensive assessment of the morphology and dynamics of cells committed to the neuronal lineage in mammalian brain primordia, we generated two transgenic mouse lines expressing a destabilized (d4) Venus controlled by regulatory elements of the Neurogenin2 (Neurog2) or Gadd45g gene. In mid-embryonic neocortical walls, expression of Neurog2-d4Venus mostly overlapped with that of Neurog2 protein, with a slightly (1 h) delayed onset. Although Neurog2-d4Venus and Gadd45g-d4Venus mice exhibited very similar labeling patterns in the ventricular zone (VZ), in Gadd45g-d4Venus mice cells could be visualized in more basal areas containing fully differentiated neurons, where Neurog2-d4Venus fluorescence was absent. Time-lapse monitoring revealed that most d4Venus+ cells in the VZ had processes extending to the apical surface; many of these cells eventually retracted their apical process and migrated basally to the subventricular zone, where neurons, as well as the intermediate neurogenic progenitors that undergo terminal neuron-producing division, could be live-monitored by d4Venus fluorescence. Some d4Venus+ VZ cells instead underwent nuclear migration to the apical surface, where they divided to generate two d4Venus+ daughter cells, suggesting that the symmetric terminal division that gives rise to neuron pairs at the apical surface can be reliably live-monitored. Similar lineage-committed cells were observed in other developing neural regions including retina, spinal cord, and cerebellum, as well as in regions of the peripheral nervous system such as dorsal root ganglia. These mouse lines will be useful for elucidating the cellular and molecular mechanisms underlying development of the mammalian nervous system.

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD 50 ) for neurovirulence in mice following intracerebral inoculation about 10 3 -fold lower than the LD 50 of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo .

Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We recently identified serine at Us3 position 147 (Ser-147) as a physiological phosphorylation site of Us3 (A. Kato, M. Tanaka, M. Yamamoto, R. Asai, T. Sata, Y. Nishiyama, and Y. Kawaguchi, J. Virol. 82:6172-6189, 2008). In the present study, we investigated the effects of phosphorylation of Us3 Ser-147 on regulation of Us3 catalytic activity in infected cells and on HSV-1 pathogenesis. Our results were as follows. (i) Only a small fraction of Us3 purified from infected cells was phosphorylated at Ser-147. (ii) Us3 phosphorylated at Ser-147 purified from infected cells had significantly higher kinase activity than Us3 not phosphorylated at Ser-147. (iii) Phosphorylation of Us3 Ser-147 in infected cells was dependent on Us3 kinase activity. (iv) Replacement of Us3 Ser-147 by alanine significantly reduced viral replication in the mouse cornea and the development of herpes stromal keratitis and periocular skin disease in mice. These results indicated that Us3 catalytic activity is tightly regulated by autophosphorylation of Ser-147 in infected cells and that regulation of Us3 activity by autophosphorylation appeared to play a critical role in viral replication in vivo and in HSV-1 pathogenesis.

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca(2+)) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca(2+) indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca(2+) indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca(2+) imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

Herpesvirus nucleocapsids assemble in the nucleus and must cross the nuclear membrane for final assembly and maturation to form infectious progeny virions in the cytoplasm. It has been proposed that nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane, and these enveloped nucleocapsids then fuse with the outer nuclear membrane to enter the cytoplasm. Little is known about the mechanism(s) for nuclear egress of herpesvirus nucleocapsids and, in particular, which, if any, cellular proteins are involved in the nuclear egress pathway. UL12 is an alkaline nuclease encoded by herpes simplex virus type 1 (HSV-1) and has been suggested to be involved in viral DNA maturation and nuclear egress of nucleocapsids. Using a live-cell imaging system to study cells infected by a recombinant HSV-1 expressing UL12 fused to a fluorescent protein, we observed the previously unreported nucleolar localization of UL12 in live infected cells and, using coimmunoprecipitation analyses, showed that UL12 formed a complex with nucleolin, a nucleolus marker, in infected cells. Knockdown of nucleolin in HSV-1-infected cells reduced capsid accumulation, as well as the amount of viral DNA resistant to staphylococcal nuclease in the cytoplasm, which represented encapsidated viral DNA, but had little effect on these viral components in the nucleus. These results indicated that nucleolin is a cellular factor required for efficient nuclear egress of HSV-1 nucleocapsids in infected cells.

We recently reported that the herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) (A. Kato, J. Arii, I. Shiratori, H. Akashi, H. Arase, and Y. Kawaguchi, J. Virol. 83:250-261, 2009; T. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller and D. C. Johnson, J. Virol. 83:3115-3126, 2009). In the studies reported here, we examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo and present data showing that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis and periocular skin disease in mice. The same effects have been reported for mice infected with a recombinant HSV-1 carrying a kinase-inactive mutant of Us3. These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically reacted with phosphorylated gB Thr-887 and used this antibody to show that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly on the cell surface of infected cells.