Immunogenomic profiles associated with response to neoadjuvant chemoradiotherapy in patients with rectal cancer.

BACKGROUND: Accumulating evidence suggests that radiotherapy success has an immune-associated component. The immunogenomic profiles associated with responses to chemoradiotherapy (CRT) were assessed in patients with locally advanced rectal cancer in this study. METHODS: CD8+ tumour-infiltrating lymphocyte (TIL) and stromal lymphocyte densities were assessed by immunohistochemistry using pretreatment biopsies from patients with advanced rectal cancer who had preoperative CRT. Whole-exome sequencing and gene expression microarray analysis were conducted to investigate the genomic properties associated with the response to CRT and CD8+ TIL density. Response to CRT was determined based on Dworak tumour regression grade (TRG); tumours with complete (TRG 4) or near-complete (TRG 3) regression were grouped as good responders, and those with TRG 1 as non-responders. RESULTS: Immunohistochemical examinations (275 patients) showed that pre-CRT CD8+ TIL density was associated with better response to CRT and improved recurrence-free survival, whereas pre-CRT stromal CD8+ cell density was not associated with either response to CRT or recurrence-free survival. Whole-exome sequencing (74 patients) showed that the numbers of single-nucleotide variations (SNVs) and neoantigens predicted from SNVs were higher in good responders than in non-responders, and these correlated positively with CD8+ TIL density (rS = 0·315 and rS = 0·334 respectively). Gene expression microarray (90 patients) showed that CD8A expression correlated positively with the expression of programmed cell death 1 (PDCD1) (rS = 0·264) and lymphocyte-activation gene 3 (LAG3) (rS = 0·507). CONCLUSION: Pre-CRT neoantigen-specific CD8+ T cell priming may be a key event in CRT responses where immune checkpoint molecules could be useful targets to enhance tumour regression.

ANTECEDENTES: Las evidencias existentes sugieren que el éxito de la radioterapia tiene un componente asociado con el sistema inmunitario. En este estudio se evaluaron los perfiles inmunogenómicos asociados con la respuesta a la quimiorradioterapia (chemoradiotherapy, CRT) en pacientes con cáncer de recto localmente avanzado. MÉTODOS: Las densidades de los linfocitos infiltrantes de tumor CD8+ (tumour-infiltrating lymphocyte, TIL) y de los linfocitos del estroma se evaluaron por inmunohistoquímicas en las biopsias antes del tratamiento de pacientes con cáncer de recto localmente avanzado que recibieron CRT preoperatoria. Se realizó secuenciación de todo el exoma, así como microarrays de expresión génica, para investigar las propiedades genómicas asociadas con la respuesta a la CRT y a la densidad de los TIL CD8+. La respuesta a la CRT se determinó según el grado de regresión del tumor de Dworak (tumour regression grade, TRG), agrupándose como buenos respondedores los casos de regresión tumoral completa (TRG4) o casi completa (TRG3) y como no respondedores, los casos de grado TRG1. RESULTADOS: Los exámenes inmunohistoquímicos (n = 275) mostraron que la densidad pre-CRT de TIL CD8+ se asoció con una mejor respuesta a la CRT y una mejor supervivencia libre de recidiva, aunque la densidad de células CD8+ del estroma previa a la CRT no se asoció con la respuesta a la CRT ni con la supervivencia libre de recidiva. La secuenciación de todo el exoma (n = 74) mostró que el número de variaciones de nucleótidos únicos (single nucleotide variations, SNVs) y los neoantígenos predichos a partir de los SNVs fueron mayores en los que respondieron bien que en los que no respondieron, y éstos se correlacionaron positivamente con la densidad de los TIL CD8+ (Spearman r = 0,315 y r = 0,334 respectivamente). Los microarrays de expresión génica (n = 90) mostraron que la expresión CD8A se correlacionó positivamente con la expresión del ligando de muerte programada-1 (r = 0,264) y con el antígeno linfocitario del gen 3 (r = 0,507). CONCLUSIÓN: La activación de células T CD8+ específicas para neoantígenos previa a la CRT puede ser un evento clave en la respuesta a la misma donde las moléculas del punto de control inmunitario podrían ser dianas útiles para intensificar la regresión del tumor.

Quantitative characterization of T-cell repertoire in allogeneic hematopoietic stem cell transplant recipients.

Allogeneic hematopoietic stem cell transplantation (HSCT) is one of curative treatment options for patients with hematologic malignancies. Although GVHD mediated by the donor's T lymphocytes remains the most challenging toxicity of allo-HSCT, graft-versus-leukemia (GVL) effect targeting leukemic cells, has an important role in affecting the overall outcome of patients with AML. Here we comprehensively characterized the TCR repertoire in patients who underwent matched donor or haplo-cord HSCT using next-generation sequencing approach. Our study defines the functional kinetics of each TCRA and TCRB clone, and changes in T-cell diversity (with identification of CDR3 sequences) and the extent of clonal expansion of certain T-cells. Using this approach, our study demonstrates that higher percentage of cord-blood cells at 30 days after transplant was correlated with higher diversity of TCR repertoire, implicating the role of cord-chimerism in enhancing immune recovery. Importantly, we found that GVHD and relapse, exclusive of each other, were correlated with lower TCR repertoire diversity and expansion of certain T-cell clones. Our results highlight novel insights into the balance between GVHD and GVL effect, suggesting that higher diversity early after transplant possibly implies lower risks of both GVHD and relapse following the HSCT transplantation.

Kinetic Interpretation of the Importance of OATP1B3 and MRP2 in Docetaxel-Induced Hematopoietic Toxicity.

Neutropenia is a lethal dose-limiting toxicity of docetaxel. Our previous report indicated that the prevalence of severe docetaxel-induced neutropenia is significantly associated with genetic polymorphisms in solute carrier organic anion transporter 1B3 (SLCO1B3) (encoding organic anion-transporting polypeptide 1B3 (OATP1B3)) and ATP-binding cassette subfamily C2 (ABCC2) (encoding multidrug-resistant-associated protein 2 (MRP2)). Therefore, we investigated their significance in docetaxel-induced neutropenia. In vitro experiments suggested their possible involvement in the hepatic uptake of docetaxel and its efflux from bone marrow cells. To further characterize a quantitative impact of OATP1B3 and MRP2 on neutropenia, we used an in silico simulation of the neutrophil count in docetaxel-treated subjects with functional changes in OATP1B3 and MRP2 in a pharmacokinetic/pharmacodynamic model. The clinically reported odds ratios for docetaxel-induced neutropenia risk were explained by the decreased function of OATP1B3 and MRP2 to 41 and 32%, respectively. These results suggest that reduced activities of OATP1B3 and MRP2 associated with systemic exposure and local accumulation in bone marrow cells, respectively, account for the docetaxel-induced neutropenia observed clinically.

Case report: dose adjustment of warfarin using genetic information and plasma concentration monitoring.

WHAT IS KNOWN AND OBJECTIVE: Carbamazepine is known to interact with warfarin. We report on a case of this interaction and on its management using the patient's genetic information. CASE SUMMARY: The case concerns a 74-year-old Japanese woman with a mood disorder and a central retinal vein occlusion. She was on therapy that included carbamazepine and had started to take warfarin. However, the patient's prothrombin time expressed as the international normalized ratio (PT-INR) was 1·40 despite taking a dose three times higher than the average. The patient's S-warfarin concentration was 0·15 µg/mL and R-warfarin was 0·52 µg/mL. Her cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex, subunit 1 (VKORC1), genotypes were *1/*1 and -1639GA, respectively. The VKORC1 genotype indicated that she would require an even higher dose. We proposed a further increase in dose and the patient's PT-INR rose to 1·99. WHAT IS NEW AND CONCLUSION: The patient required a high warfarin dose because of the VKORC1 genotype, and induction of CYP2C9 by carbamazepine. We improved the patient's pharmacotherapy based on her genetic information.

CYP2D6 genotype and adjuvant tamoxifen: meta-analysis of heterogeneous study populations.

The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor-positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease-free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow-up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

Ethnic differences between Japanese and Caucasians in the expression levels of mRNAs for CYP3A4, CYP3A5 and CYP3A7: lack of co-regulation of the expression of CYP3A in Japanese livers.

Using a newly developed real-time reverse transcriptase-polymerase chain reaction method, mRNAs were quantitated for CYP3A4, CYP3A5 and CYP3A7 in adult livers from 24 Japanese and 24 Caucasian subjects to elucidate the potential ethnic differences in the expression levels of human cytochrome P450 (CYP) 3As. The expression level of CYP3A4 mRNA in Japanese livers (n = 24) was approximately three times higher than that in Caucasian livers (n = 24, p < 0.001). The mean level of CYP3A5 mRNA was approximately twice higher in Japanese (n = 9) than in Caucasians (n = 5) heterozygous for the CYP3A5 *1 allele (p = 0.057). The CYP3A7 mRNA level was twice higher in Japanese (n = 24) than in Caucasians (n = 22) carrying the CYP3A7 *1A/ *1A genotype (p = 0.042). The level of CYP3A4 mRNA did not correlate with those of CYP3A5 (r = 0.044, n = 24) or CYP3A7 (r = 0.21, n = 24) mRNAs in Japanese livers in contrast to co-regulatory expression of CYP3A4, CYP3A5 and CYP3A7 in Caucasian livers. The results indicate that there are ethnic differences in the expression levels of adult liver CYP3A mRNAs between Japanese and Caucasians, and that the mechanism(s) regulating the hepatic CYP3A expression may be different between these ethnic groups.

Attenuation of a field Sendai virus isolate through egg-passages is associated with an impediment of viral genome replication in mouse respiratory cells.

We investigated the mechanisms responsible for attenuation of mouse pathogenicity of Sendai virus (SeV) through passages in eggs. A highly virulent clone, E0, derived from the field SeV Hamamatsu strain, was successively passaged in hen's eggs. Analysis of the mouse lethal dose 50% (MLD50) of virus clones obtained from the viruses at egg-passages 1, 15, 30 and 50 demonstrated that attenuation of E0 by egg-passage occurred due to the gradual appearance of and replacement by virus variants possessing higher MLD50. Comparison of viral replication in the mouse lung and mouse pathogenicity with the representative SeV clones, E0, E15c12, E30c12 and E50c19, obtained from the respective egg-passages revealed that the low pathogenicity of the egg-passaged clones was due to poor multi-cycle viral replication in the lung. Furthermore, MLD50s of the SeV clones were found to be negatively correlated with the replication capability in primary mouse pulmonary epithelial (MPE) cells; the egg-passaged clones with more attenuated phenotypes showed lower replication capability in MPE cells. In the MPE cells infected with the SeV clones at m.o.i. 10, however, viral protein and mRNA syntheses of the egg-passaged clones were enhanced or comparable to those of the parental E0 clone at 1 day and 2 days post infection (p.i.) but decreased more rapidly thereafter. In contrast, viral genome synthesis of the egg-passaged clones in the cells at 2 days p.i. was several times lower than that of E0. These results strongly suggest that attenuation of a virulent field SeV strain by egg-passage occurs due to the appearance and selection of virus variants possessing poor propagation capacity in mouse respiratory epithelial cells, which is caused primarily by an impediment of viral genome replication.

Conserved and non-conserved regions in the Sendai virus genome: evolution of a gene possessing overlapping reading frames.

We have sequenced the entire genome of a virulent field isolate of Sendai virus, the Hamamatsu strain, and compared the sequence with that of a distant related strain, the Z strain. Calculation of synonymous and non-synonymous (amino acid changing) nucleotide substitutions revealed regions where changes were permissive and non-permissive, and the experimentally determined functional region were found to be conserved, showing that important regions for function were conserved during evolution. In the cistron-overlapping regions in the P gene, one reading frame was conserved, whereas the other overlapping frame was flexible. The priority of one frame could be a strategy for evolution of an overlapping gene of RNA viruses. We found that the carboxyl two thirds of the C protein was conserved over the amino-terminal one third, possessing priority to the overlapping P polypeptide. This suggests that the carboxyl two thirds of the C protein have a functional importance. We also found a highly variable region between the L coding frame and the 5' trailer sequence. The relevance of these findings to actual viral replication should be clarified in the future.

Involvement of the zinc-binding capacity of Sendai virus V protein in viral pathogenesis.

The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C(341)S and V-C(365)R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV V(DeltaC), which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

Role of primary constitutive phosphorylation of Sendai virus P and V proteins in viral replication and pathogenesis.

Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis.

Double-layered membrane vesicles released from mammalian cells infected with Sendai virus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) was reported to form vesicles on the cell surface and subsequently to be released into the cultured medium when expressed from cDNA by virus vectors. To further investigate VSV M activity, we generated a recombinant Sendai virus (SeV) expressing the VSV M protein (SeV-M(VSV)). When cells were infected with SeV-M(VSV), VSV M was found abundantly in the culture medium. Electron microscopy demonstrated the budding of two-membraned vesicles (>/= 0.8 microm in diameter) from the infected cells. The outer membrane of the vesicle was derived from the plasma membrane and the inner one possibly derived from the membrane of an intracellular vesicle. Immuno-gold labeling showed that VSV M was exclusively located in a double-layered region. The released membranes were divided into three parts: the VSV M vesicles with SeV F and HN glycoproteins, SeV particles, and vesicles associated with the cytosolic components. The last abundantly contained phosphorylated SeV matrix (M) protein, which is not released in a usual SeV infection. Furthermore the VSV M protein expressed without using a virus vector was efficiently released into the culture medium. These results suggest that the VSV M protein has a budding activity per se and that SeV proteins are passively involved in the release of VSV M.

Sendai virus gene start signals are not equivalent in reinitiation capacity: moderation at the fusion protein gene.

In paramyxovirus transcription, viral RNA polymerase synthesizes each monocistronic mRNA by recognizing the gene start (S) and end (E) signals flanking each gene. These signal sequences are well conserved in the virus family; nevertheless, they do exhibit some variations even within a virus species. In Sendai virus (SeV) Z strain, the E signals are identical for all six genes but there are four (N, P/M/HN, F, and L) different S signals with one or two nucleotide variations. The significance of these variations for in vitro and in vivo replication has been unknown. We addressed this issue by SeV reverse genetics. The luciferase gene was placed between the N and P gene so that recombinant SeVs expressed luciferase under the control of each of the four different S signals. The S signal for the F gene was found to drive a lower level of transcription than that of the other three, which exhibited comparable reinitiation capacities. The polar attenuation of SeV transcription thus appeared to be not linear but biphasic. Then, a mutant SeV whose F gene S signal was replaced with that used for the P, M, and HN genes was created, and its replication capability was examined. The mutant produced a larger amount of F protein and downstream gene-encoded proteins and replicated faster than wild-type SeV in cultured cells and in embryonated eggs. Compared with the wild type, the mutant virus also replicated faster in mice and was more virulent, requiring a dose 20 times lower to kill 50% of mice. On the other hand, the unique F start sequence as well as the other start sequences are perfectly conserved in all SeV isolates sequenced to date, including highly virulent fresh isolates as well as egg-adapted strains, with a virulence several magnitudes lower than that of the fresh isolates. This moderation of transcription at the F gene may therefore be relevant to viral fitness in nature.

Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication.

Sendai virus (SeV) is an enveloped virus with a negative sense genome RNA of about 15.3 kb. We previously established a system to recover an infectious virus entirely from SeV cDNA and illustrated the feasibility of using SeV as a novel expression vector. Here, we have attempted to insert a series of foreign genes into SeV of different lengths to learn how far SeV can accommodate extra genes and how the length of inserted genes affects viral replication in cells cultured in vitro and in the natural host, mice. We show that a gene up to 3.2 kb can be inserted and efficiently expressed and that the replication speed as well as the final virus titers in cell culture are proportionally reduced as the inserted gene length increases. In vivo, such a size-dependent effect was not very clear but a remarkably attenuated replication and pathogenicity were generally seen. Our data further confirmed reinforcement of foreign gene expression in vitro from the V(-) version of SeV in which the accessory V gene had been knocked out. Based on these results, we discuss the utility of SeV vector in terms of both efficiency and safety.

Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases.

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the -4, -2 and -1 positions upstream of the cleavage site were essential, and that at the -5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.

Endogenous protease-dependent replication of human influenza viruses in two MDCK cell lines.

Multi-cycle replication and plaque formation of influenza A and B viruses and cleavage activation of their hemagglutinin (HA) by an endogenous protease(s) were examined in two MDCK cell lines, MDCK(-) and MDCK(+). No exogenous trypsin was required for multi-cycle replication and plaque formation of all the influenza A viruses tested in the MDCK(+) cell, while those of the viruses in the MDCK(-) cell were completely trypsin-dependent. In both cell lines, on the other hand, influenza B viruses grew well in the absence of trypsin. The capability of multiple replication and plaque formation of the influenza viruses correlated with cleavage of the HA precursor (HA0) to HA1 and HA2, indicating that both cell lines express an HA activating endoprotease(s); that of the MDCK(+) cell activates the HA of influenza A and B viruses, and that of the MDCK(-) cell does only the HA of influenza B virus. Furthermore, the protease of the MDCK(+) cell was strongly suggested to be present on the cell surface and a serine protease. The MDCK(+) cell would be useful for isolation of influenza viruses from clinical specimens and for screening of protease inhibitors for anti-influenza virus drugs.

Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis.

BACKGROUND: The P/C mRNA of Sendai virus (SeV), a prototypic member of the family Paramyxoviridae in the Mononegavirales superfamily comprising a large number of nonsegmented negative strand RNA viruses, encodes a nested set of accessory proteins, C', C, Y1 and Y2, referred to collectively as C proteins, initiating, respectively, at ACG/81 and AUGs/114, 183, 201 in the +1 frame relative to the ORF of phospho (P) protein, the smaller subunit of RNA polymerase. Among them, C is the major species expressed in infected cells at a molar ratio which is several-fold higher than the other three. However, their function has remained an enigma. It has not even been established whether or not the C proteins are essential for viral replication. Many other viruses in Mononegavirales encode C-like proteins, but their roles also remain to be defined. RESULTS: By taking advantage of a recently developed reverse genetics system to recover infectious SeV from cDNA, we created mutants in which C protein frames were variously silenced. C/C'(-) viruses which did not express C and C', but did express Y1 and Y2, were severely attenuated in replication in tissue culture cells of various species and tissues, as well as in embryonated chicken eggs. More notably, they were almost totally incapable of growing productively in--and hence nonpathogenic for mice--the natural host. Both gene expression and genome replication appeared to be impaired in C/C'(-) viruses. Additionally silencing the Y1 and Y2 expression was also possible, and a critically impaired but viable clone, the 4C(-) virus, was isolated which expressed none of the four C proteins. CONCLUSION: SeV C proteins are categorically nonessential gene products, but greatly contribute to full replication capability in vitro and are indispensable for in vivo multiplication and pathogenesis. This study represents the first comprehensive functional assessment of the accessary C protein for Mononegavirales.

Importance of the cysteine-rich carboxyl-terminal half of V protein for Sendai virus pathogenesis.

The Sendai virus V protein is a nonstructural trans-frame protein whose cysteine-rich C-terminal half is fused to the acidic N-terminal half of the P protein via mRNA editing. We recently created a mutant by disrupting the editing motif, which is devoid of mRNA editing and hence unable to produce the V protein, and demonstrated that this V(-) virus replicated normally or even faster with augmented gene expression and cytopathogenicity in cells in vitro, but was strongly attenuated in pathogenicity for mice (A. Kato, K. Kiyotani, Y. Sakai, T. Yoshida, and Y. Nagai, EMBO J. 16:578-587, 1997). Thus, although categorized as a nonessential protein, the V protein appeared to encode a luxury function required for the viral in vivo pathogenesis. Here, we created another version of a V-deficient mutant, VdeltaC, encoding only the N-terminal half but not the V-specific C-terminal half, by introducing a stop codon in the trans-V frame, and then we compared its in vitro and in vivo phenotypes with those of the V(-) and wild-type viruses. The VdeltaC virus was found to be similar to the wild-type virus in vitro with no augmented gene expression and cytopathogenicity, but in vivo, it resembled the V(-) virus, displaying a similarly attenuated phenotype. Thus, the pathogenicity determinant in the V protein was mapped to the C-terminal half. The N-terminal half was likely sufficient to confer normal (wild-type) in vitro phenotypes.

Phosphorylation of the Sendai virus M protein is not essential for virus replication either in vitro or in vivo.

A large proportion of intracellular Sendai virus (SeV) M proteins is phosphorylated, but in mature virions the M protein is not phosphorylated or dephosphorylated. Phosphorylated M protein in cells is bound to the cytoskeletal components more firmly than unphosphorylated M protein. Thus it has been hypothesized that M protein phosphorylation plays an important role in the virus life cycle, especially in the step of maturation. Here, a transient expression-mutation experiment of the M gene demonstrated that a change of the Ser residue at the 70th position from the N-terminus to Ala (S70A) totally abolished M protein phosphorylation, strongly suggesting that this residue is phosphorylated. The mutated M gene was then placed in the corresponding region in the cDNA plasmid which generates a full-length antigenome SeV RNA, and a mutant SeV M-S70A was successfully recovered from the cDNA. This mutant virus was indeed defective in M protein phosphorylation but did not differ at all from the wild-type SeV recovered from the parental cDNA either in the replication kinetics and plaque morphology in cultured cells or in in vivo replication and pathogenicity for mice. We thus concluded that no phosphorylation of the M protein was required for SeV replication either in vitro or in vivo.

The paramyxovirus, Sendai virus, V protein encodes a luxury function required for viral pathogenesis.

The Sendai virus (SeV) V protein is characterized by the unique cysteine-rich domain in its carboxy-terminal half which is fused to the amino-terminal half of the P protein, but its function has remained enigmatic. The V protein-directing mRNA is generated by a remarkable process known as mRNA editing involving the pseudotemplated addition of a single G residue at a specific septinucleotide locus in the P gene, whereas the unedited exact copy encodes the P protein. Here, we introduced two nucleotide changes in the septinucleotide motif (UUUUCCC to UUCUUCC) in a full-length SeV cDNA and were able to recover a virus from the cDNA, which was devoid of mRNA editing and hence unable to synthesize the V protein. Compared with the parental wild-type virus with regard to gene expression, replication and cytopathogenicity in various cell lines in vitro, the V(-) virus was found to be either potentiated or comparable but never attenuated. The V(-) virus, however, showed markedly attenuated in vivo replication capacity in and pathogenicity for mice. Thus, though categorized as a nonessential gene product, SeV V protein encodes a luxury function required for in vivo pathogenicity.

Protection of mice from respiratory Sendai virus infections by recombinant vaccinia viruses.

Mechanisms of protection of mice from Sendai virus, which is exclusively pneumotropic and causes a typical respiratory disease, by immunization with recombinant vaccinia viruses (RVVs) were investigated. Although the RVV carrying a hemagglutinin-neuraminidase gene of Sendai virus (Vac-HN) propagated in the noses and lungs of mice by either intranasal (i.n.) or intraperitoneal (i.p.) inoculation, no vaccinia virus antigens were detected in the mucosal layer of upper and lower airways of the i.p.-inoculated mice. The mice immunized i.n. with Vac-HN or Vac-F (the RVV carrying a fusion protein gene of Sendai virus) demonstrated the strong resistance to Sendai virus challenge both in the lung and in the nose, whereas the i.p.-immunized mice showed almost no resistance in the nose but showed a partial resistance in the lung. Titration of Sendai virus-specific antibodies in the nasal wash (NW), bronchoalveolar lavage (BAL), and serum collected from the Vac-F-immunized mice showed that the NW from the i.n.-immunized mice contained immunoglobulin A (IgA) antibodies but no IgG and the BAL from the mice contained both IgA and IgG antibodies. On the other hand, neither IgA nor IgG antibodies were detected in the NW from the i.p.-immunized mice and only IgG antibodies were detected in the BAL, although both i.n.- and i.p.-immunized mice exhibited similar levels of serum IgG, IgA, and neutralizing antibodies. The resistance to Sendai virus in the noses of i.n.-immunized mice could be abrogated by the intranasal instillation of anti-mouse IgA but not of anti-IgG antiserum, while the resistance in the lung was not significantly abrogated by such treatments. These results demonstrate that IgA is a major mediator for the immunity against Sendai virus induced by the RVVs and IgG is a supplementary one, especially in the lung, and that the RVV should be intranasally inoculated to induce an efficient mucosal immunity even if it has a pantropic nature.