Protease activation mutants of sendai virus. Activation of biological properties by specific proteases.

A new class of Sendai virus mutants (pa mutants) is described that exhibit altered specificities with respect to protease activation of infectivity and altered host range. Sendai virus requires proteolytic cleavage of a virion glycoprotein (F₀ to F) in order to be infective. Wild-type virus can be activated in vitro by treatment with trypsin, but not chymotrypsin or elastase, or in vivo by addition of trypsin to cells, e.g., MDBK, which lack activating protease. Mutants have been isolated that are activated by chymotrypsin (pa-c mutants) or elastase (pa-e mutants). Some mutants are no longer activated by trypsin, and these mutants have lost the ability to undergo multiple-cycle replication in the embryonated chick egg unless chymotrypsin or elastase is added to the mutants also activate hemolysis. These findings with pa mutants support the previous conclusion based on results with wild type virus, that a host-dependent cleavage of the F₀ protein is required for the infectivity of Sendai virus and for activation of hemolyzing and cell-fusing activities. The results obtained indicate that the host range and tissue tropism of Sendai virus are determined at least in part by the availability of the appropriate protease required for activation of infectivity.     

Evidence of proteolytic activation of Sendai virus in mouse lung

A device was made to analyze the pneumotropism of Sendai virus in mouse. Minced lung blocks were prepared from the mouse intranasally infected with Sendai virus for 2 hours and cultured in a CO2 incubator. This culture system provided a suitable in vitro model of Sendai virus infection in mice in terms of the distribution of the viral antigens and histopathological findings. The progeny virus recovered from the lung culture was already activated and was accompanied by the cleavage of F glycoprotein into F1 and F2. This fact demonstrates that the activating mechanism is reversed in the lung culture as found in vivo infection of mouse lung. The viral activation and the cleavage of F glycoprotein were simultaneously inhibited by tosyllysylchloromethylketone, leupeptin, soybean trypsin inhibitor and antipain, but not by tosylamidophenylethylchloromethyl-ketone, chymostatin, pepstatin, iodoacetamide, phenylmethylsulfonylfluoride and p-chloromercuribenzoate. These results show that the activating enzyme of Sendai virus found in the lung culture was similar to trypsin. The existence of the activating enzyme may support the replication of Sendai virus in mouse lung in multiple-step and also result in the lung pathology.     

A comparison in germ-free mice of the pathogenesis of Sendai virus and mouse pneumonia virus infections

The poathogenesis of Sendai virus and pneumonia virus of mice (PVM) was studied using the immunoperoxidase technique on paraffin lung sections. The pathology of Sendai virus corresponds to that of a bronchopneumonia, with virus demonstrated by immunoperoxidase in the bronchial epithelium, and sometimes in macrophages, for a period of 2--9 days post-infection. Pneumonia virus of mice produces an interstitial pneumonia with virus demonstrated in the bronchial epithelium but also in the alveolar walls and alveolar macrophages. This virus can be demonstrated between 2 and 7 days post-infection. This technique was used to demonstrate PVM in the case of a natural outbreak of this disease and may eventually become a routine technique for the screening of lung tissue for respiratory viruses.     

Mutations in Sendai virus variant F1-R that correlate with plaque formation in the absence of trypsin

With the emergence of new viruses, such as the SARS virus and the avian influenza virus, the importance of investigations on the genetic basis of viral infections becomes clear. Sendai virus causes a localized respiratory tract infection in rodents, while a mutant, F1-R, causes a systemic infection. It has been suggested that two determinants are responsible for the systemic infection caused by F1-R [Okada et al (1998) Arch Virol 143:2343–2352]. The primary determinant of the pantropism is the enhanced proteolytic cleavability of the fusion (F) protein of F1-R, which allows the virus to undergo multiple rounds of replication in many different organs, whereas wild-type virus can only undergo multiple rounds of replication in the lungs. The enhanced cleavability of F1-R F was previously attributed to an amino acid change at F115 that is adjacent to the cleavage site at amino acid 116. Secondly, wild-type virus buds only from the apical domain of bronchial epithelium, releasing virus into the lumen of the respiratory tract, whereas F1-R buds from both apical and basolateral domains. Thus, virus is released into the basement membrane where it can easily gain access to the bloodstream for dissemination. The microtubule disruption is attributed to two amino acid differences in M protein. To confirm that the F and M gene mutations described above are solely responsible for the phenotypic differences seen in wld-type versus F1-R infections, reverse genetics was used to construct recombinant Sendai viruses with various combinations of the mutations found in the M and F genes of F1-R. Plaque assays were performed with or without trypsin addition. A recombinant virus containing all F1-R M and F mutations formed plaques in LLC-MK2 cells and underwent multiple cycles of replication without trypsin addition. To clarify which mutation(s) are necessary for plaque formation, plaque assays were done using other recombinant viruses. A virus with only the F115 change, which was previously thought to be the only change important for plaque formation of F 1-R F, did not confer upon the virus the ability to form plaques without the addition of trypsin. Another virus with the F115 and both M changes gave the same result. Therefore, more than one mutation in the F gene contributes to the ability of F1-R to form plaques without trypsin addition.     

Persistence of pneumonia virus of mice and Sendai virus in germ-free (nu/nu) mice.

The pathogenicity and persistence of pneumonia virus of mice (PVM) and Sendai virus has been studied using germ-free nu/nu mice. PVM was found to infect cells of the bronchial epithelium (and the alveolar wall) of the lungs of germ-free nu/nu mice using the immunoperoxidase technique. The virus was located in the bronchial epithelium for 11 days before elimination, but persisted in the alveolar wall for the duration of the experiment (20 days). After Day 10 a humoral antibody response to PVM was observed which persisted, although at a low level (1 in 40), by haemagglutination-inhibition (HI) testing. Sendai virus in nu/nu mice also infected cells of the bronchial epithelium and this persisted for the duration of the experiment (27 days). The persistence of virus in the bronchial epithelium in relation to lack of humoral antibody is discussed with reference to local secretory antibody production, especially since this does not occur with PVM.     

Inhibitory effect of pulmonary surfactant on Sendai virus infection in rat lungs

Summary Intranasal infection of rats with active (infectious) Sendai virus enhances secretion of tryptase Clara, a Sendai virus-activating protease, into the bronchial lumen by Clara cells of the bronchial epitheliums, and inversely suppresses secretion of pulmonary surfactant, an inhibitor of the protease, into the lumen [Kido H et al. (1993) FEBS Lett 322: 115–119]. A trypsin-resistant mutant, TR-2, showed similar effects, although its replication was restricted to a single cycle in the lungs. In contrast, neither nonactive (noninfectious) wild-type virus possessing receptor-binding activity and lacking envelope fusion activity nor UV-inactivated virus retaining receptor binding and envelope fusion activities altered the mode of secretions. These results indicate that viral replication is required for producing a condition in the bronchial lumen for proteolytic activation of progeny virus, thereby infection is extended to a fatal pneumonia. On the other hand, intranasal administration of infected rats with pulmonary surfactant suppressed activation of progeny virus and pathological changes in the lungs, suggesting a therapeutic use of pulmonary surfactant for influenza pneumonia.     

Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of Sendai virus

The glycoproteins of Sendai virus have been isolated by a procedure involving extraction with the nonionic detergent Triton X-100 and affinity chromatography on fetuin-Sepharose. The largest Sendai virus glycoprotein (MW ~69,000) possesses both hemagglutinating and neuraminidase activities, and has been designated HN.Virions grown in MDBK cells or in the allantoic sac of the chick embryo contain similar amounts of the HN glycoprotein, but differ in their content of the other glycoproteins. Virions grown in MDBK cells contain a large amount of a glycoprotein designated F₀ (MW ~65,000). This glycoprotein is a precursor of a smaller virion glycoprotein, F (MW ~53,000) which is present in only small amount in MDBK cellgrown virions. F₀ can be cleaved to yield F by treatment of virions with trypsin in vitro. Sendai virions grown in the chick embryo lack the precursor protein F₀, but contain a large amount of F; in this case, proteolytic cleavage of F₀ to yield F occurs in ovo.The Sendai virions grown in MDBK cells, which are deficient in the small glycoprotein F, lack hemolyzing and cell-fusing activities and cannot infect MDBK cells. Virions grown in the chick embryo, which contain much F, possess both these activities and can infect MDBK cells as well as the chick embryo. MDBK cell-grown virions acquire hemolyzing and cell-fusing activities and become infective for MDBK cells when the precursor glycoprotein F₀ is cleaved in vitro to yield F.The results indicate that the small glycoprotein of paramyxoviruses is biologically active and is involved in virus-induced hemolysis, cell fusion, and the initiation of infection. The precise mechanism by which this glycoprotein participates in these reactions remains to be determined, but is now amenable to experimentation. The precursor of this glycoprotein is biologically inactive, but is incorporated into virions grown in some host cells; it may be activated by proteolytic cleavage either in vivo or in vitro. The present results provide a biochemical basis for previously observed host-dependent variation in infectivity, and in hemolysis and cell-fusion induced by paramyxoviruses.     

Fusion of sendai virus with liposome depends on only F protein, but not HN protein

Sendai virus is able to fuse with liposomes even without virus receptors. To determine the roles of envelope protein, hemagglutinin-neuraminidase (HN) and fusion (F) protein, in Sendai virus-liposome fusion, we treated the virus with proteases and examined its fusion with liposomes and the conditions of HN and F protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analysis showed that the virus treated with 150 units/ml of trypsin, which inactivated selectively hemolysis activity, maintained intact HN, F and partially digested F (32 kDa) protein, while virus treated with 15,000 units/ml of trypsin, which inactivated both hemolysis and neuraminidase activity, had only a 15-kDa digested HN protein and completely digested F protein. The former fused with liposomes, but the latter did not. In the virus treated with chymotrypsin, which lost both hemolysis and neuraminidase activity, F protein was intact, while HN protein was degraded to 15 kDa; in this case the virus fused with liposomes. As the virus with 15-kDa HN protein fused with liposomes and that with 20-kDa protein did not, HN protein does not appear to play any role in virus-liposome fusion. The virus that fused with liposomes had intact F protein. We conclude that Sendai virus-liposome fusion is strongly dependent on the presence of intact F protein, but not HN protein.     

Comparison of protective effects of serum antibody on respiratory and systemic infection of Sendai virus in mice

Summary The protective effects of the passive administration of convalescent serum from mice infected with Sendai virus were evaluated in mice challenged intranasally with wild-type and a pantropic variant (F1-R) of Sendai virus. Adoptive transfer of the serum efficiently prevented F1-R from infecting the systemic organs, but it failed to protect the mice from infections of the respiratory tracts by either virus. Virus replication in nasal turbinates was not diminished while infection in the lung was suppressed sufficiently for the infected mice to survive the infection. These findings suggest that serum antibody is less effective for the protection against viral infections on the surface of the respiratory tract, but it is effective for inhibition of spread of the virus into the systemic organs.     

Chemiluminescence: an early event in the interaction of Sendai and influenza viruses with mouse spleen cells. I. The role of the envelope glycoproteins in the stimulation of chemiluminescence

On infection with Sendai virus, non-adherent mouse spleen cells emit a burst of chemiluminescence (CL) starting within a few seconds and peaking at 6–8 min postinfection. The biological reactions leading to CL are not known in mouse spleen cells, but in phagocytic cells are believed to be correlated with the generation of unstable oxygen species by the cells (e.g., H₂0₂, 0₂^?, OH·, singlet oxygen). In this paper, we have investigated the mechanism of CL induction by the virus. Envelope particles, possessing the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins stimulated CL, suggesting that the biochemical reactions leading to light emission are triggered by the interaction of the envelope “spike” glycoproteins with the cell surface. The individual contributions of HN and F to CL stimulation were investigated by removing F from egg-grown virus and by using Sendai virus (grown in MDBK cells) which possesses F₀, the biologically inactive precursor of F. Both viral preparations still induced CL. However, CL was reduced and the peak of light emission shifted from 6–8 to 2.5 min postinfection. In MDBK cell-grown Sendai virus, cleavage of F₀ into F resulted in the increase of CL and shift back of the peak CL to 6–8 min postinfection. These results suggest that the bulk of light emission by the spleen cells is correlated with the action of the F protein. In addition, HN correlates with CL in the initial period following the addition of the virus to the spleen cell suspension, while F is important for the subsequent further increase in light emission. The mechanism of CL induction by the F glycoprotein was investigated using egg-grown nonhemolytic “early harvest” and hemolytic “late harvest” Sendai virus, respectively. “Early harvest” virus, known to possess F and to have fusion activity, was less active in CL induction than “late harvest” Sendai virus which expresses both the fusion and hemolytic activities. This suggests that F stimulates CL by a mechanism related to hemolysis, rather than by envelope-cell membrane fusion. In addition, we show that influenza virus (strain A/RI/5-) also induces CL. The kinetics and extent of CL induction by this virus were similar to those induced by Sendai virus lacking the fusion protein and by nonhemolytic Sendai virus possessing F. As with Sendai virus, Pronase treatment resulted in the loss of CL stimulation while uv-inactivation of the virus did not affect its CL-inducing activity, suggesting that influenza virus also triggers CL by a mechanism involving the envelope glycoproteins.     

Characterization of a pantropic variant of Sendai virus derived from a host range mutant

A variant (F1-R) was isolated from a temperature-sensitive host range mutant (ts-f1) of Sendai virus. F1-R was no longer temperature-sensitive but it retained the host range phenotype. Unlike wild-type virus, F1-R and ts-f1 undergo multiple cycles of replication in several cell lines in the absence of trypsin. This was attributed to proteolytic activation of the fusion (F) glycoprotein of the host range mutants, in cell nonpermissive to wild-type virus. In mice infected intranasally the variant F1-R caused a generalized infection. This was shown by immunohistology and with infectious virus being recovered from several organs whereas infection with wild-type virus was restricted to the lung. These observations indicate that the pantropic property of F1-R is the result of proteolytic activation of the virus by ubiquitous proteases. Nucleotide sequence analyses revealed that ts-f1 and F1-R differed from the wild-type virus by mutations at the region of the cleavage site of F and at the glycosylation site of the F2 subunit. The findings indicated that these mutations are responsible for the increased cleavability of the F protein of ts-f1 and F1-R and therefore are important determinants for the pantropism of F1-R.     

Determinants of pantropism of the F1-R mutant of Sendai virus: specific mutations involved are in the F and M genes

Summary.  Mutations in the fusion, F, protein of Sendai virus resulting in increased cleavability by ubiquitous host protease(s), and mutations in the matrix, M, protein resulting in bipolar budding, are both important determinants for the systemic infection in mice caused by the protease activating pantropic mutant, F1-R. Several mutants of Sendai virus (BY, BF, and KD-M) with phenotypes of bipolar budding and/or increased cleavability of F protein were isolated. Genomic RNA sequence analysis of the F and M genes of the mutants revealed that several deduced amino acids in the F and M proteins were different from those of F1-R, T-5 (a revertant of F1-R), and wild-type viruses. The BF and KD-M mutants that budded bipolarly and were also activated by ubiquitous proteases were examined for replication in tissue culture cells and in mice. All of the mutants exhibited multiple-step replication in MDCK, MDBK, and LLC-MK2 cells without trypsin, but formed plaques only in MDCK cells. One of the mutants, designated KD-52M, was similar to F1-R in that it formed plaques in all three cell lines without addition of exogenous protease. However, none of the mutant viruses, including KD-52M, caused a systemic infection in mice. The mutated M protein of F1-R enhances the disruption of microtubles. However, none of the mutants with a bipolar budding phenotype (BY, BF, and KD-M), disrupted the microtubules to the same extent as F1-R. All of these mutants had mutations in the M protein that were different from those found in F1-R. Taken together, these results suggest that mutations at Ser115 to Pro in the F protein and at Asp 128 to Gly and Ile210 to Thr in the M protein of F1-R are the mutations specifically required for the systemic infection caused by F1-R. Received May 18, 1998 Accepted July 6, 1998     

Pneumotropic revertants derived from a pantropic mutant, F1-R, of Sendai virus.

Revertants were isolated from the protease activation mutant of Sendai virus, F1-R, which causes a systemic infection in mice. The fusion (F) glycoprotein of F1-R is susceptible to activation cleavage by ubiquitous cellular proteases and is thus responsible for pantropism in mice (Tashiro et al., 1988. Virology 165, 577-583). The revertants regained several phenotypes of wild-type virus; they required exogenous trypsin for activation of the F protein in cell cultures and in nonpulmonary mouse tissues and they were exclusively pneumotropic in mice. On the other hand, phenotypes of F1-R that remained unchanged by the revertants were bipolar budding in polarized epithelial cells, enhanced electrophoretic migration of the matrix protein, and the lack of a glycosylation site in the F2 subunit of the F protein. Comparative RNA sequence analysis of the F gene of the revertants revealed that the reduced cleavability of the F protein of the revertants was the result of the predicted single amino acid reversion (Pro to Ser) at residue 115 adjacent to the cleavage site. Thus the sequence at the cleavage site of the revertants was Ser-Lys compared with Pro-Lys for F1-R and Ser-Arg for wild-type virus. The results indicate that enhanced cleavability of the glycoprotein, a feature often associated with multiple basic residues within the cleavage site of paramyxovirus F proteins and influenza virus hemagglutinins, can also be determined by a single basic amino acid following proline. Additionally, the revertants were less susceptible to the activator for wild-type virus present in mouse lungs and less pathogenic for this organ than wild-type virus. These results provide further evidence that proteolytic activation of the F protein by host proteases is the primary determinant for organ tropism and pathogenicity of Sendai virus in mice. One of the revertants was also temperature sensitive (ts); the ts lesion in the nucleoprotein gene was identical to that found in ts-f1, the ts host range mutant from which F1-R was derived.     

Immediate protection of mice from lethal wild-type Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, possessing homologous interfering capacity

Protection of mice from lethal Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, which was isolated from a carrier culture, was studied. HVJpi had a strong interfering capacity with the replication of virulent wild-type virus in LLCMK2 cells. When a high dose of HVJpi (3.0 x 10(7) CIU) was inoculated intranasally into mice, the mice showed neither illness nor lung lesions but gained significant resistance against the challenge of virulent wild-type virus (18 LD50) immediately after inoculation. In contrast, the mice inoculated with a lower dose of HVJpi (8.2 x 10(5) CIU) did not show the immediate resistance but became immune several days after inoculation. Time courses of the virus replication in the lung revealed that the replication of wild-type virus was strongly suppressed to about 1/1000 by the simultaneous infection with a high dose of HVJpi, thus resulting in minimizing the lung lesions and survival of all the mice infected. Neither interferon nor natural killer cells appeared to play a major role in the immediate immune status by HVJpi, since no difference was observed in protection of mice simultaneously infected with wild-type virus and HVJpi in spite of pretreatment of the mice with anti-interferon and anti-asialo GM1 antibodies as compared with that of the untreated doubly infected mice. On the other hand, it was suggested by analysis of viral polypeptides synthesized in the lung of infected mice by Western blotting that the early stage of replication of wild-type virus in the lung was inhibited mainly by the interfering capacity of HVJpi. These results indicate that HVJpi is an unique virus mutant which is capable of protecting mice from lethal Sendai virus infections by its interfering capacity immediately after inoculation and then by the induction of virus-specific immune responses.     

Significance of basolateral domain of polarized MDCK cells for Sendai virus-induced cell fusion

Summary Fusion (fusion from within) of polarized MDCK monolayer cells grown on porous membranes was examined after infection with Sendai viruses. Wild-type virus, that buds at the apical membrane domain, did not induce cell fusion even when the F glycoprotein expressed at the apical domain was activated with trypsin. On the other hand, a protease activation mutant, F 1-R, with F protein in the activated form and that buds bipolarly at the apical and basolateral domains, caused syncytia formation in the absence of exogenous protease. Anti-Sendai virus antibodies added to the basolateral side, but not at the apical side, inhibited cell fusion induced by F 1-R. In addition, T-9, a mutant with bipolar budding phenotype of F 1-R but with an uncleavable F protein phenotype like wild-type virus, induced cell fusion exclusively when trypsin was added to the basolateral medium. By electron microscopy, cell-to-cell fusion was shown to occur at the lateral domain of the plasma membrane. These results indicate that in addition to proteolytic activation of the F protein, basolateral expression of Sendai virus envelope glycoproteins is required to induce cell fusion.     

Parainfluenza Virus Sendai Infection in Macrophages, Ependyma, Choroid Plexus, Vascular Endothelium and Respiratory Tract of Mice

Immunofluorescent observations showed that after intranasal instillation of parainfluenza 1 (Sendai) virus into adult mice, infection is confined to the epithelial lining of the larger airways. Alveolar macrophages were not significantly involved, although they could be infected in vitro. In suckling mice, the infection was more acutely lethal and extended into the terminal air spaces. The intranasal susceptibility of adult mice was not reproducibly affected by treatment with potent antithymocyte serum, and there were no obvious pathogenic effects when heterologous antiserum was instilled intranasally into infected mice. Peritoneal macrophages were infected by intraperitoneally injected Sendai virus, with production of a highly viscous peritoneal exudate. Kupffer cells of the liver and endothelial cells in large veins and auricles were infected by intravenously injected virus. When injected intracerebrally, Sendai virus infected ependyma and choroid plexus epithelium. Adult mice often survived, in spite of ependymal destruction and changes in ventricular morphology. Astrocytes were activated but not infected.     

Loss on serial passage of rhesus monkey kidney cells of proteolytic activity required for Sendai virus activation.

Primary and secondary cultures of rhesus monkey kidney cells supported multiple-cycle replication of Sendai virus, but later passages lost this ability, and this was reflected in decreased plaque formation. Multiple-cycle replication also did not occur in LLC-MK2 cells, a continuous line of RMK cells. Failure of replication in serially passed cells was correlated with a decrease in proteolytic cleavage of a viral surface glycoprotein (Fo), and the ability of cells to support multiple-cycle replication and plaque formation could be restored by the addition of trypsin (0.3 microgram/ml) to the overlay medium. The use of wild-type virus, which requires trypsin, and protease activation mutants that require chymotrypsin or elastase for activation has provided evidence that the activating protease supplied by primary or secondary cells has trypsin-like activity. Inactive virus, with uncleaved Fo glycoprotein, absorbed to primary or secondary cells but did not infect them, even though such cells possess the enzyme that is capable of cleaving the Fo glycoprotein of virus synthesized in these cells. The inability of these cells to activate adsorbed virus indicates that the activating protease that they possess is inacessible to adsorbed virus, although it can act on the Fo glycoprotein during virus maturation in these cells. These data provide a biochemical explanation for the failure of later passages of a cell strain or a continuous cell line to support the replication of a paramyxovirus.     

Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage

With strain Ulster of Newcastle disease virus, two precursor glycoproteins, HN₀ and F₀, were identified; these are converted by proteolytic cleavage into glycoproteins HN and F, respectively. Purified virions containing predominantly glycoproteins HN₀ and F₀ together with a small amount of HN are not hemolytic and have reduced levels of hemagglutinating and neuraminidase activity and of infectivity. After in vitro treatment with the appropriate proteolytic enzymes, biological activities are fully expressed in these particles. The precursor glycoprotein HN₀ was isolated and found to be largely devoid of hemagglutinating and neuraminidase activities. High levels of both activities were present, however, when this material was subjected to proteolytic cleavage. These observations demonstrate that cleavage is a precondition for the biological activity not only of glycoprotein F but also of glycoprotein HN. There is a striking difference between glycoproteins HN₀ and F₀ with repsect to their susceptibility to proteolytic enzymes. Cleavage and activation of HN₀ can be accomplished by a variety of proteases, such as chymotrypsin, elastase, thermolysin, and trypsin. In contrast, F₀ shows a specific requirement for trypsin.     

Budding site of Sendai virus in polarized epithelial cells is one of the determinants for tropism and pathogenicity in mice.

Wild-type Sendai virus fusion (F) glycoprotein requires trypsin or a trypsin-like protease for cleavage-activation in vitro and in vivo, respectively. The virus is pneumotropic in mice and buds at the apical domain of bronchial epithelial cells. On the other hand, the F protein of the protease-activation host range mutant, F1-R, is cleaved by ubiquitous proteases present in different cell lines and in various organs of mice. F1-R causes a systemic infection in mice and the mutant buds bipolarly at the apical and basolateral domains of infected epithelial cells. The enhanced cleavability of the F protein of F1-R has been shown to be a primary determinant for pantropism. Additionally, it has been postulated that bipolar budding of F1-R is required for the systemic spread of the virus and it has been attributed to mutations in the matrix (M) protein of F1-R (Tashiro et al., Virology 184, 227-234, 1991). In this study protease-activation mutants (KD series) were isolated from wild-type virus. They were revealed to bud at the apical domain, and the F protein was cleaved by ubiquitous proteases in mouse organs. The KD mutants were exclusively pneumotropic in mice following intranasal infection, whereas they caused a generalized infection when inoculated directly into the circulatory system. Comparative nucleotide sequence analysis of the F gene of the KD mutants revealed that the deduced amino acid substitutions responsible for enhanced cleavability of the F protein occurred removed from the cleavage site. Mutations were not at all found in the M gene of the KD mutants analyzed, in support of the role of the M protein of F1-R and of a revertant T-9 derived from the latter in bipolar budding. These results suggest that bipolar budding is necessary for the systemic spread of F1-R from the lungs and that apical budding by wild-type virus and the KD mutants leads to respiratory infections. Differential budding at the primary target of infection, in addition to the cleavage-activation of the F protein in mouse organs, is therefore also a determinant for tropism and pathogenicity of Sendai virus in mice.     

Masking of the contribution of V protein to Sendai virus pathogenesis in an infection model with a highly virulent field isolate

Sendai virus V protein is not essential for virus replication in cultured cells but is essential for efficient virus replication and pathogenesis in mice, indicating that the V protein has a luxury function to facilitate virus propagation in mice. This was discovered in the Z strain, an egg-adapted avirulent laboratory strain. In the present study, we reexamined the function of Sendai virus V protein by generating a V-knockout Sendai virus derived from the Hamamatsu strain, a virulent field isolate, which is an appropriate model for studying the natural course of Sendai virus infection in mice. We unexpectedly found that the V-knockout virus propagated efficiently in mice and was as virulent as the wild-type virus. Switching of the functionally important V unique region demonstrated that this region of the Hamamatsu strain was also functional in a Z strain background. It thus appears that the V protein is nonsense in a field isolate of Sendai virus. However, the V protein was required for virus growth and pathogenesis of the Hamamatsu strain in mice when the virulence of the virus was attenuated by introducing mutations that had been found in an egg-adapted, avirulent virus. The V protein therefore seems to be potentially functional in the highly virulent Hamamatsu strain and to be prominent if virus replication is restricted.