Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59

The murine coronavirus [murine hepatitis virus (MHV)] is limited to infection of susceptible mice and murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on murine and non-murine cells.     

Changes of the receptor-binding properties of influenza B virus B/Victoria/504/2000 during adaptation in chicken eggs

Selection of high-growth virus variants of strain B/Victoria/504/2000 by serial passage in eggs resulted in three amino acid substitutions, G141E, R162M, and D196Y, in the vicinity of the receptor-binding pocket of viral hemagglutinin. Virus variants containing the identified amino acid substitutions, individually or in various combinations, were constructed using reverse genetics and analyzed for their receptor-binding properties using glycan microarray platform. Three different patterns of virus binding were revealed. A low-growth virus variant, corresponding to the original egg-derived virus B/Victoria/504/2000 prior to acquisition of amino acid changes G141E, R162M, and D196Y, had a clear preference for the oligosaccharide chains terminated with α2-6-linked sialic acid with very weak binding of the glycans terminated with α2-3-linked sialic acid. Amino acid substitutions R162M and D196Y had similar effects, resulting in viruses that bound with high efficiency almost all terminally sialylated glycans represented on the array regardless of the type of glycosidic linkage. In contrast, substitution of G141E alone, or in combinations with the other two amino acid substitutions, significantly restricted virus glycan-binding capabilities. All virus variants possessing this substitution lost the ability to bind glycans with α2-6 glycosidic linkage as well as most of the glycans with α2-3 glycosidic linkage. Linear penta- and heptasaccharide chains represented at the non-reducing end by α2-3 sialylated Type-II motif (LacNAc) were the only structures bound with high affinity by the virus variants with G141E substitution. In all cases when the effects on virus binding of individual amino acid substitutions differed, the effect of R162M was subordinate to the effect of either G141E or D196Y.     

Generation of the influenza B viruses with improved growth phenotype by substitution of specific amino acids of hemagglutinin

Variability in growth characteristics of influenza B viruses remains a serious limitation in the manufacture of inactivated influenza vaccines. Currently, serial passage in eggs is the strategy used in most instances for selection of high growth virus variants. In previous studies we found that adaptation of the strain B/Victoria/504/2000 to high growth in eggs was associated with changes only in hemagglutinin (HA). The high growth phenotype was associated with acquisition of either two (R162M and D196Y) or three (G141E, R162M and D196Y) amino acid (AA) substitutions, predicted to be near the receptor-binding domain of HA. In the present study we analyzed, using reverse genetics, the contribution to virus growth of each of these AA substitutions and determined their effect on antigenic properties. We found that G141E and R162M were most favorable for virus growth; however, only R162M could improve virus growth without antigenic alteration. Substitution D196Y had least effect on virus growth but substantially altered antigenic properties. Additional virus variants with AA substitutions at positions 126, 129, 137 and 141 were generated and characterized. The AA changes advantageous for growth of B/Victoria/504/2000 were also tested in the context of the HA of the B/Beijing/184/93, a virus with stable low-growth phenotype. All of the tested AA substitutions improved the replicative capabilities of the corresponding viruses, but only N126D and K129E had no effect on antigenicity. The results of our studies demonstrate that introduction of specific AA substitutions into viral HA can improve viral replicative efficiency while preserving the original antigenic properties.     

Fabry disease: 45 novel mutations in the alpha-galactosidase A gene causing the classical phenotype

The nature of the molecular lesions in the alpha-galactosidase A (alpha-Gal A) gene causing Fabry disease was determined in 50 unrelated families with the classic phenotype of this X-linked recessive lysosomal storage disease. Genomic DNA was isolated from affected males or obligate carrier females, and the entire alpha-Gal A coding region as well as the flanking and intronic sequences were analyzed by PCR amplification and automated sequencing. Forty-five new mutations were identified including 38 single base substitutions (32 missense and four nonsense) and nine gene rearrangements: MIR, M42T, G43D, G43V, H46Y, F50C, L68F, G132R, T141I, Y152X, K168R, G183S, V199M, P205R, Y207S, Q221X, C223R, C223Y, D234Y, G271C, A288P, P293A, R301G, I303N, I317T, E341D, P362L, R363C, R363H, G373D, I384N, T385P, Q396X, E398K, S401X, P409A, g7325insC, g7384del13, g8341delG, g8391del4/ins3, g10511delTAGT, g10704delACAG, g11019insG, g11021insG, and g11048delAGG. In the remaining five Fabry families, four previously reported mutations were detected (W81X, R112C, g11011delTC, and g11050delGAG) of which the R112C substitution was found in two families who were unrelated by haplotyping. These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing the classical Fabry disease phenotype, and permit precise carrier detection and prenatal diagnosis in these families.     

MHV S peplomer protein expressed by a recombinant vaccinia virus vector exhibits IgG Fc-receptor activity.

We have previously shown that cells infected with mouse hepatitis virus (MHV) bind rabbit, mouse, and rat IgG by the Fc portion of the IgG molecule. This Fc-binding activity appeared to be mediated by the MHV S protein. S protein could also be precipitated from MHV-infected cells by a monoclonal antibody directed against the murine Fc gamma receptor (Fc gamma R). To prove definitively that the S protein mediates Fc-binding activity, we have expressed the MHV S protein utilizing recombinant vaccinia viruses. The anti-Fc gamma R monoclonal antibody, 2.4G2, precipitated recombinant S protein in cells of murine, human, and rabbit origin. Since the anti-Fc receptor monoclonal antibody does not react with human and rabbit Fc receptors these results demonstrate that the epitope recognized by this antibody is carried on the MHV S protein and is not murine in origin. Examination of various MHV isolates and escape mutants failed to identify the precise sequences in S responsible for the molecular mimicry of the murine Fc gamma R. These data are consistent with the hypothesis that a previously identified region of similarity between the S protein and the Fc gamma R mediates this activity. The Fc binding activity of S was expressed on the cell surface, since MHV-JHM-infected cells, but not uninfected cells, formed rosettes with anti-sheep red blood cell (SRBC) antibody-coated SRBC. The anti-Fc gamma R monoclonal antibody neutralized MHV-JHM and inhibited syncytium formation induced by the MHV S protein.     

Missense mutations in the extracellular domain of the human neural cell adhesion molecule L1 reduce neurite outgrowth of murine cerebellar neurons

Mutations in L1CAM, the gene encoding the transmembrane multifunctional neuronal adhesion molecule L1, are associated with neurodevelopmental disorders including X-linked hydrocephalus and mental retardation. Some amino acid substitutions in various extracellular domains of L1 are known to affect posttranslational processing of the protein or its homophilic and heterophilic interactions. It is largely unknown, however, how these mutations result in neurodevelopmental disturbances and whether the effects of mutations on neurodevelopment can be modeled in vitro. We stably expressed full-length human wild type L1 and the known pathogenic missense mutations I179S, R184W, Y194C, and C264Y in NIH-3T3 cells. L1 protein synthesis, glycosylation pattern, and subcellular localization were analyzed. Neurite outgrowth of primary murine cerebellar neurons was measured after 23 hrs of co-cultivation using transfected NIH-3T3 cells as substrate. Like wild type L1, L1 protein with I179S or Y194C mutations was localized on the surface of the transfected substrate cells, but this was not the case with R184W or C264Y mutations. All four mutations were associated with reduced stimulation of neurite outgrowth. Measurement of neurite outgrowth on transfected substrate cells may be a suitable model for studying neurodevelopmental disturbances.     

Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

Isolation of a murine hepatitis virus from Swiss mice treated with antilymphocyte serum

Summary In an attempt to transmit feline malignant lymphoma to mice, a murine hepatitis virus (MHV) was accidentally recovered from conventionally reared Swiss mice receiving prolonged treatment with antilymphocyte serum. In these mice, the virus did not require concomitant infection withEperythrozoon coccoides to produce disease. Tests for antibodies against a variety of viruses were performed on serum from control colony mice and mice that recovered from experimental infection as well as on serum pooled from vaccinated and non-vaccinated mice challenged with the recovered agent. Antibodies to the MHV complement fixing (CF) antigen(s) were demonstrated in only the last mentioned serum. Mice harbouring a hepatitis virus may thus be tolerant to their CF antigen(s) in a situation analogous to oncornavirus infection of their natural hosts. The liver pathology was that of a confluent focal-type necrosis resembling that produced by certain other MHV strains. We have labelled this newly isolated virus MHV (Swiss-Cape Town), abbreviated to MHV (S-CT).     

Characterization of pertussis toxin analogs containing mutations in B-oligomer subunits.

The S2, S3, and S4 subunit genes of pertussis toxin (PT) from Bordetella pertussis were subjected to site-directed mutagenesis, and the resultant PT analogs were assayed for altered biological properties. PT analogs S2(T91,R92,N93) delta and S2(Y102A,Y103A) exhibited reduced binding to fetuin. Several PT analogs with mutations in the S2, S3, or S4 subunit showed reduced in vitro toxicity, as measured in the Chinese hamster ovary (CHO) cell clustering assay. In particular, PT analogs S3(Y82A) and S3(I91,Y92,K93) delta retained 10% or less residual toxicity. These mutants also exhibited significantly lower mitogenic and hemagglutinating activities and reduced in vivo activities, as measured by the histamine sensitization and leukocytosis assays. The S4(K54A,K57A) PT analog had significantly reduced CHO cell clustering activity, though other biological activities remained unaffected. PT analogs S1(E129G)/S3(Y82A) and S1(E129G)/S3(I91,Y92,K93) delta displayed a cumulative effect of the S1 and S3 mutations for both in vitro and in vivo toxic activities. These PT analogs, as well as S1(R9K,E129G)/S3(K82A) and S1(R9K,E129G)/S3(I91,Y92,K93) delta, still expressed an epitope which elicits a neutralizing antitoxin antibody and were protective in the mouse intracerebral challenge test. Recombinant pertussis vaccines based on PT analogs with detoxifying mutations in multiple subunits may thus represent the next generation of improved whooping cough vaccines.     

Multiple recombination sites at the 5'-end of murine coronavirus RNA

Mouse hepatitis virus (MHV), a murine coronavirus, contains a nonsegmented RNA genome. We have previously shown that MHV could undergo RNA-RNA recombination in crosses between temperature-sensitive mutants and wild-type viruses at a very high frequency (S. Makino, J.G. Keck, S.A. Stohlman, and M.M.C. Lai (1986) J. Virol. 57, 729-737). To better define the mechanism of RNA recombination, we have performed additional crosses involving different sets of MHV strains. Three or possibly four classes of recombinants were isolated. Recombinants in the first class, which are similar to the ones previously reported, contain a single crossover in either gene A or B, which are the 5'-most genes. The second class of recombinants contain double crossovers in gene A. The third class of recombinants have crossovers within the leader sequence located at the 5'-end of the genome. The crossover sites of the third class have been located between 35 and 60 nucleotides from the 5'-end of the leader RNA. One of these recombinants has double crossovers within the short region comprising the leader sequences. Finally, we describe one recombinant which may contain a triple crossover. The presence of so many recombination sites within the 5'-end of the genome of murine coronaviruses confirms that RNA recombination is a frequent event during MHV replication and is consistent with our proposed model of "copy-choice" recombination in which RNA replication occurs in a discontinuous and nonprocessive manner.     

Functional interaction between the N- and C-terminal domains of murine leukemia virus surface envelope protein

A series of murine leukemia viruses (MuLVs) with chimeric envelope proteins (Env) was generated to map functional interactions between the N- and the C-terminal domains of surface proteins (SU). All these chimeras have the 4070A amphotropic receptor-binding region flanked by various lengths of Moloney ecotropic N- and C-terminal Env. A charged residue, E49 (E16 on the mature protein), was identified at the N-terminals of Moloney MuLV SU that is important for the interaction with the C-terminal domain of the SU. The region that interacts with E49 was localized between junction 4 (R265 of M-MuLV Env) and junction 6 (L374 of M-MuLV Env) of SU. Sequencing the viable chimeric Env virus populations identified residues within the SU protein that improved the replication kinetics of the input chimeric Env viruses. Mutations in the C-domain of SU (G387E/R, L435I, L442P) were found to improve chimera IV4, which displayed a delayed onset of replication. The replication of AE6, containing a chimeric junction in the SU C-terminus, was improved by mutations in the N-domain (N40H, E80K), the proline-rich region (Q252R), or the transmembrane protein (L538N). Altogether, these observations provide insights into the structural elements required for Env function.     

Acute GB virus B infection of marmosets is accompanied by mutations in the NS5A protein

GBV-B, a member of the Flaviviridae family of viruses, is the virus most closely related to HCV, and GBV-B infection in tamarin monkeys might represent a valuable surrogate animal model of HCV infection. In the current study, GBV-B was successfully transmitted to two marmosets (Callithrix jaccus). The infection resulted in viremia of 14- and 17-week duration, respectively, and was accompanied by elevation of isocitrate dehydrogenase activity. These data confirm that marmosets might represent an attractive model for GBV-B infection. The sequence of GBV-B NS5A, which was previously reported to have one of the highest mutation rates during infection in tamarins, was determined for viruses recovered from the inoculum and from marmoset blood samples obtained at weeks 1, 8, and 14 post inoculation in one marmoset and at weeks 2, 8, and 17 post inoculation in the other marmoset. In both animals, we detected four substitutions (R1945K, K2052G, F2196L, and G2268E), in the virus recovered immediately before viral clearance. Interestingly, two of these mutations (F2196L and G2268E) were described recently for viruses recovered from persistently infected tamarins. Appearance of these mutations presumably reflects a mechanism of immune escape rather than adaptation of the virus to a new host.     

Mechanisms of pathogenicity in human MSH2 missense mutants

The human mismatch repair (MMR) gene MSH2 is the second most frequently mutated hereditary nonpolyposis colorectal cancer (HNPCC) susceptibility locus. Given that missense mutations account for 17% of all identified alterations in this gene, the study of their pathogenicity is of increasing importance. Previously, we showed that pathogenic MSH2 missense mutations typically impaired the repair activity of the protein. In this study, we took advantage of its crystal structure and attempted to correlate the mismatch binding and ATP-catalyzed mismatch release activities with the location of 18 nontruncating MSH2 mutations. We observed that the MMR-deficient mutations situated in the amino-terminal connector and lever domains of MSH2 (V161D, G162R, G164R, L173P, L187P, C333Y, and D603N) affected protein stability, whereas mutations in the ATPase domain (A636P, G674A, C697F, I745_I746del, and E749 K) mainly caused defects in mismatch binding or release. Of the MMR-proficient variants, four (T33P, A272 V, G322D, and V923E) showed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) protein, while two variants (N127S and A834 T) showed no defects in the assays. Similar to our biochemical data, the mutations that affected protein stability were associated with an absence of the protein in tumors in immunohistochemical (IHC) analyses. In contrast, the protein with the mutation E749 K, which abrogates MMR but not protein stability, is well expressed in tumors. In conclusion, pathogenic missense mutations in MSH2 may interfere with different mechanisms that tend to cluster in separate protein domains with varying effects on protein stability, which could be taken into account when interpreting IHC data.     

Attenuation of Recombinant Vesicular Stomatitis Viruses Encoding Mutant Glycoproteins Demonstrate a Critical Role for Maintaining a High pH Threshold for Membrane Fusion in Viral Fitness

A plasmid-based recovery system was used to generate four unique vesicular stomatitis virus (VSV) mutants that encode glycoproteins (G proteins) with single or double amino acid substitutions in two conserved acidic residues adjacent to the putative G protein fusion domain. Previously we demonstrated that three of the mutant G proteins (D137-L, E139-L, and DE-SS) have slightly reduced pH thresholds for membrane fusion activity. In this report we show that even though the viruses encoding D137-L, E139-L, and DE-SS were recovered with high efficiency, these mutants were attenuated for growth in cell culture. Plaque formation was significantly delayed with these mutants and the plaques were smaller and more diffuse than those produced by wild-type VSV. In addition, cells infected with these mutants produced approximately 5- to 10-fold less infectious virus than cells infected with a similarly recovered VSV encoding the wild-type G protein. Using R18-labeled virus we found that the mutant G proteins had approximately 50% of the fusion activity of wild-type G at pH 6.3 and only 75% activity at pH 5.8. We also show that the mutant viruses were more sensitive to chloroquine inhibition of infection than either wild-type VSV or the mutant E139-T, which has a fusion phenotype similar to wild-type G protein. Reduced fusion activity and attenuation of infectivity was not due to differences in the amount of G protein incorporated into virions, nor to differences in the amount of virus binding to cells at physiological pH. Although infectivity was assayed at neutral pH, we observed an increase in virus binding with both mutant and wild-type virions as the pH was lowered, and the increase in binding occurred near the pH threshold for membrane fusion activity. From these data we propose a model in which VSV entry involves an increase in virus binding to the inner leaflet of the endosomal membrane during endosome acidification. Concomitant with this higher affinity binding, G protein becomes primed to initiate fusion of the viral envelope with the endosomal membrane. Viruses with mutations that delay the onset of increased binding and fusion lag behind wild-type VSV in their ability to initiate a productive infection, potentially because the location within the cytoplasm where these viruses ultimately fuse is not optimal for either virus uncoating or replication of the viral genome.     

Antigenic relationship between five isolates of murine gammaherpesvirus analysed with monoclonal antibodies

Summary.  A panel of six monoclonal antibodies (mAbs) specific for murine gammaherpesvirus (MHV) was used for analysis of the antigenic relationship between five MHV-isolates (MHV 68, MHV 72, MHV 76, MHV 78, MHV S). Two mAbs raised against MHV 72 and four raised against MHV S were used in the study. Antibody-virus interactions were tested using immunochemical (ELISA, Western blot, immunofluorescence) and biological (virus-neutralization) assays. Immunoanalysis by ELISA showed a close antigenic relationship between the five viruses, nevertheless, some antigenic individuality of the isolate MHV S was observed. This isolate originated from a geographically distinct area in Czechia relative to the other four isolates from Slovakia. In Western blot analysis, antibodies to MHV 72 recognized viral antigens with the relative molecular mass about 116 000. Of four mAbs against MHV S, only two reacted with denatured viral antigen in Western blot and showed specificity for the 50–55 000 protein. These findings suggested that both isolates, besides of minor antigenic variability, could differ also in immunodominant proteins. Mabs to MHV S exhibited much stronger virus-neutralizing potency than mAbs to MHV 72, indicating thus that the 50–55 000 antigen might be more relevant for the infectivity of MHV-virus. Immunofluorescence with mAbs allowed specific localization of antigens in virus-infected VERO cells. Received July 22, 2002; accepted February 6, 2003 Published online April 9, 2003     

X-ray structure of the hemagglutinin of a potential H3 avian progenitor of the 1968 Hong Kong pandemic influenza virus

We have determined the structure of the HA of an avian influenza virus, A/duck/Ukraine/63, a member of the same antigenic subtype, H3, as the virus that caused the 1968 Hong Kong influenza pandemic, and a possible progenitor of the pandemic virus. We find that structurally significant differences between the avian and the human HAs are restricted to the receptor-binding site particularly the substitutions Q226L and G228S that cause the site to open and residues within it to rearrange, including the conserved residues Y98, W153, and H183. We have also analyzed complexes formed by the HA with sialopentasaccharides in which the terminal sialic acid is in either alpha2,3- or alpha2,6-linkage to galactose. Comparing the structures of complexes in which an alpha2,3-linked receptor analog is bound to the H3 avian HA or to an H5 avian HA leads to the suggestion that all avian influenza HAs bind to their preferred alpha2,3-linked receptors similarly, with the analog in a trans conformation about the glycosidic linkage. We find that alpha2,6-linked analogs are bound by both human and avian HAs in a cis conformation, and that the incompatibility of an alpha2,6-linked receptor with the alpha2,3-linkage-specific H3 avian HA-binding site is partially resolved by a small change in the position and orientation of the sialic acid. We discuss our results in relation to the mechanism of transfer of influenza viruses between species.     

Partial deletion in the spike endodomain of mouse hepatitis virus decreases the cytopathic effect but maintains foreign protein expression in infected cells

Mouse hepatitis virus (MHV) produces a series of subgenomic RNAs for viral protein expression. As a prototype coronavirus, MHV has been explored extensively and is often used to express foreign proteins. Previously, a 13-residue deletion in the MHV spike (S) protein endodomain was found to reduce syncytium formation dramatically while inhibiting virus replication slightly. In this study, the effects of the S mutation on MHV infectivity and foreign protein expression were further examined in rat or mouse L2, NIH/3T3 and Neuro-2a cells. The replacement of the MHV 2a/haemagglutinin-esterase gene with a membrane-anchored protein hook (HK) and replacement of gene 4 with EGFP did not change the adaptability and cytopathology of recombinant viruses in these cells. However, the cytopathic effect of the recombinants with the partial S deletion was reduced significantly in these cells. The replication and foreign protein expression of the S-mutated recombinants were found to be more efficient in L2 cells than in Neuro-2a and NIH/3T3 cells. Meanwhile, the distribution patterns of HK and EGFP expressed by the recombinant viruses were similar to those in cells transfected with a eukaryotic expression vector. These results suggest that the partial deletion in the S endodomain may increase the usefulness of MHV as a viral vector by attenuation and maintaining foreign protein expression.