A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity

Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu(333) remained unchanged after five mouse passages, an Asn(194)-->Lys(194) mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn(194) with Lys(194) in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn(194)-->Lys(194) mutation does not arise when Asn(194) is exchanged with Ser(194) (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.     

A single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with the ability to cause growth hormone deficiency syndrome.

Persistent infection of C3H/St mice with certain strains of lymphocytic choriomeningitis virus (LCMV) causes a growth hormone (GH) deficiency syndrome (GHDS) manifested as growth retardation and hypoglycemia. Infected mice show high levels of viral replication in the GH-producing cells in the anterior pituitary leading to decreased synthesis of GH mRNA and protein despite the absence of detectable virus-induced cell structural damage. Virus clones isolated from the GHDS-negative LCMV WE strain can cause the disease, while others cannot. The genetic basis of this phenotypic difference is a nucleotide substitution resulting in a single amino acid difference in the viral glycoprotein. Reassortant studies indicate that the single amino acid substitution (Ser-153 to Phe) is sufficient to allow infection of the GH-producing cells and cause GHDS. These results show that a single change in the genome can affect viral pathogenicity by altering the tropism of the virus.     

A single amino acid change determines persistence of a chimeric Theiler's virus.

The DA strain of Theiler's virus persists in the central nervous system of mice and causes chronic inflammation and demyelination. On the other hand, the GDVII strain causes an acute encephalitis and does not persist in surviving animals. Series of recombinants between infectious cDNA clones of the genomes of DA and GDVII viruses have been constructed. The analysis of the phenotypes of the recombinant viruses has shown that determinants of persistence and demyelination are present in the capsid proteins of DA virus. Chimeric viruses constructed by the different research groups gave consistent results, with one exception. Chimeras GD1B-2A/DAFL3 and GD1B-2C/DAFL3, which contain part of capsid protein VP2, capsid proteins VP3 and VP1, and different portions of P2 of GDVII in a DA background, were able to persist and cause demyelination. Chimera R4, whose genetic map is identical to that of GD1B-2A/DAFL3, was not. After exchanging the viral chimeras between laboratories and verifying each other's observations, new chimeras were generated in order to explain this difference. Here we report that the discrepancy can be attributed to a single amino acid difference in the sequence of the capsid protein VP2 of the two parental DA strains. DAFL3 (University of Chicago) and the chimeras derived from it, GD1B-2A/DAFL3 and GD1B-2C/DAFL3, contain a Lys at position 141, while TMDA (Institut Pasteur) and R4, the chimera derived from it, contain an Asn in that position. This amino acid is located at the tip of the EF loop, on the rim of the depression spanning the twofold axis of the capsid. These results show that a single amino acid change can confer the ability to persist and demyelinate to a chimeric Theiler's virus, and they pinpoint a region of the viral capsid that is important for this phenotype.     

汉坦病毒囊膜糖蛋白G1基因重组腺病毒载体的构建及其在Vero E6细胞中的表达

拟构建汉坦病毒Gl基因重组腺病毒载体并在VeroE6细胞中表达,为汉坦病毒基因疫苗的研究提供实验基础。PCR法从含汉坦病毒-76118株M基因的M56质粒扩增糖蛋白G1基因片段,利用穿梭质粒pShuttle,将其克隆入Adeno—X病毒DNA,获得重组腺病毒DNA,转染HEK293细胞,包装、扩增后得到汉坦病毒Gl基因重组腺病毒原种,感染VetoE6细胞,用IFA法和ELISA法检测表达产物。得到了含汉坦病毒G1基因的重组腺病毒,其滴度约为10^11pfu／ml,感染VeroE6细胞后检测到汉坦病毒糖蛋白G1的表达。     

A single amino acid change in the E2 glycoprotein of Sindbis virus confers neurovirulence by altering an early step of virus replication.

Amino acid changes in the envelope glycoproteins of Sindbis virus have been linked to neurovirulence; however, the molecular mechanisms by which these amino acid changes alter neurovirulence are not known. Recombinant-virus studies have mapped an important determinant of neurovirulence in adult mice to a single amino acid change, glutamine to histidine, at position 55 of the E2 glycoprotein (P. C. Tucker, E. G. Strauss, R. J. Kuhn, J. H. Strauss, and D. E. Griffin, J. Virol. 67:4605-4610, 1993). To investigate how histidine confers neurovirulence, we examined the various stages of the virus life cycle in neural (N18) and nonneural (BHK) cells. In BHK cells, recombinant viruses 633 (E255Q) and TE (E255H) replicated similarly. In contrast, in N18 neuroblastoma cells, TE established infection more efficiently, replicated faster, and achieved higher rates of virus release than did 633. Viral structural protein synthesis was similar in 633- and TE-infected BHK cells, while in N18 cells, structural protein synthesis was detected only in TE-infected cells at 6 h and remained higher for at least 16 h postinfection. Viral RNA synthesis was initiated more rapidly and was up to fivefold greater in TE- versus 633-infected N18 cells. Taken together with other data demonstrating minimal effects on virus binding and entry (P. C. Tucker, S. H. Lee, N. Bui, D. Martinie, and D. E. Griffin, J. Virol. 71:6106-6112, 1997), these data suggest that E2 position 55 plays an important role at early stages of infection of neural cells, thereby facilitating neurovirulence.     

Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55(Gag) elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.     

A single amino acid in nonstructural protein NS4B confers virulence to dengue virus in AG129 mice through enhancement of viral RNA synthesis

Dengue (DEN) is a mosquito-borne viral disease that has become an increasing economic and health burden for the tropical and subtropical world. The lack of an appropriate animal model of DEN has greatly impeded the study of its pathogenesis and the development of vaccines/antivirals. We recently reported a DEN virus 2 (DENV-2) strain (D2Y98P) that lethally infects immunocompromised AG129 mice, resulting in organ damage or dysfunction and increased vascular permeability, hallmarks of severe DEN in patients (G. K. Tan et al., PLoS Negl. Trop. Dis. 4:e672, 2010). Here we report the identification of one critical virulence determinant of strain D2Y98P. By mutagenesis, we showed that a Phe-to-Leu alteration at amino acid position 52 in nonstructural protein NS4B completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Conversely, a Leu-to-Phe alteration at position 52 of NS4B in nonvirulent DENV-2 strain TSV01 led to 80% lethality and increased viremia. The NS4B(Phe52) viruses displayed enhanced RNA synthesis in mammalian cells but not in mosquito cells. The increased viral RNA synthesis was independent of the ability of NS4B to interfere with the host type I interferon response. Overall, our results demonstrate that Phe at position 52 in NS4B confers virulence in mice on two independent DENV-2 strains through enhancement of viral RNA synthesis. In addition to providing further insights into the functional role of NS4B protein, our findings further support a direct relationship between viral loads and DEN pathogenesis in vivo, consistent with observations in DEN patients.     

The Purkinje cell degeneration 5J mutation is a single amino acid insertion that destabilizes Nna1 protein

In the mouse, Purkinje cell degeneration (pcd) is a recessive mutation characterized by degeneration of cerebellar Purkinje cells, retinal photoreceptors, olfactory bulb mitral neurons, and certain thalamic neurons, and is accompanied by defective spermatogenesis. Previous studies of pcd have led to the identification of Nna1 as the causal gene; however, how loss of Nna1 function results in neurodegeneration remains unresolved. One useful approach for establishing which functional domains of a protein underlie a recessive phenotype has been to determine the genetic basis of the various alleles at the locus of interest. Because none of the pcd alleles analyzed at the time of the identification of Nna1 provided insight into the molecular basis of Nna1 loss-of-function, we obtained a recent pcd remutation—pcd^5J, and after determining that its phenotype is comparable to existing pcd severe alleles, we sought its genetic basis by sequencing Nna1. In this article we report that pcd^5J results from the insertion of a single GAC triplet encoding an aspartic acid residue at position 775 of Nna1. Although this insertion does not affect Nna1 expression at the RNA level, Nna1^pcd-5J protein expression is markedly decreased. Pulse-chase experiments reveal that the aspartic acid insertion dramatically destabilizes Nna1^pcd-5J protein, accounting for the observation that pcd^5J is a severe allele. The presence of a readily detectable genetic mutation in pcd^5J confirms that Nna1 loss-of-function alone underlies the broad pcd phenotype and will facilitate further studies of how Nna1 loss-of-function produces neurodegeneration and defective spermatogenesis in pcd mice.     

Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence.

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.     

汉坦病毒包膜糖蛋白G2重组腺病毒的构建及其在Hela细胞中的表达

本研究旨在构建可表达汉坦病毒(HTNV)糖蛋白G2的重组腺病毒。应用PCR方法扩增G2编码基因,经T/A克隆、测序鉴定后再亚克隆到腺病毒shuttle载体pAd5-CMV中并用磷酸钙沉淀法分别将携带G2编码基因的重组腺病毒shuttle载体与携带报告基因eGFP的腺病毒骨架质粒共转染HEK293细胞,包装、扩增、纯化后得到携带HTNV糖蛋白G2编码基因的重组腺病毒;用重组腺病毒感染Hela细胞并收获蛋白,间接免疫荧光、Western blotting检测蛋白表达。经酶切鉴定表明已成功构建了携带G2基因的重组腺病毒载体;RT-PCR鉴定表明目的基因能够在感染重组腺病毒的Hela细胞中转录;荧光显微镜观察重组腺病毒感染的Hela细胞,可见报告基因eGFP的表达;间接免疫荧光法和Western blotting均证实表达产物可被抗G2单克隆抗体所识别,表明糖蛋白G2在感染细胞中得到了表达。本研究成功构建了可表达HTNV包膜糖蛋白G2的重组腺病毒,转染宿主细胞可稳定表达目的蛋白,为HTNV糖蛋白G2的结晶、结构解析研究以及新型汉坦病毒疫苗的研制奠定了基础。     

Pathogenicity in mice of herpes simplex virus type 2 mutants unable to express glycoprotein C.

Herpes simplex virus type 2 (HSV-2) mutants that were unable to express glycoprotein C (gC-2) were isolated. Deletions were made in a cloned copy of the gC-2 gene, and recombinant viruses containing these deletions were screened by using an immunoreactive plaque selection protocol. The viruses did not display a syncytial phenotype. Intravaginal inoculation of BALB/cJ mice with one of the HSV-2 gC-2- viruses produced local inflammation followed by a lethal spread of the viral infection into the nervous system in a manner identical to that produced by parental HSV-2 strain 333. Similarly, intracerebral inoculation of DBA-2 mice with the gC-2- virus produced a lethal neurological disease paralleling that caused by HSV-2 strain 333. These results indicate that gC-2 is not required for the spread of HSV-2 infections in mice.     

Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change.

A neutralization-resistant variant of human immunodeficiency virus type 1 (HIV-1) that emerged during in vitro propagation of the virus in the presence of neutralizing serum from an infected individual has been described. A threonine-for-alanine substitution at position 582 in the gp41 transmembrane envelope glycoprotein of the variant virus was responsible for the neutralization-resistant phenotype (M.S. Reitz, Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff, Cell 54:57-63, 1988). The mutant virus also exhibited reduced sensitivity to neutralization by 30% of HIV-1-positive sera that neutralized the parental virus, suggesting that a significant fraction of the neutralizing activity within these sera can be affected by the amino acid change in gp41 (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. Robert-Guroff, J. Virol. 64:3240-3248, 1990). It is shown here that the change of alanine 582 to threonine specifically confers resistance to neutralizing by antibodies directed against both groups of discontinuous, conserved epitopes related to the CD4 binding site on the gp120 exterior envelope glycoprotein. Only minor differences in binding of these antibodies to wild-type and mutant envelope glycoproteins were observed. Thus, the antigenic structure of gp120 can be subtly affected by an amino acid change in gp41, with important consequences for sensitivity to neutralization.     

Serologic type conversion of a herpes simplex virus type 1 (HSV-1) to an HSV-2 epitope caused by a single amino acid substitution in glycoprotein C.

A monoclonal antibody to herpes simplex virus type 2 glycoprotein C (gC-2) did not recognize wild-type herpes simplex virus type 1 gC (gC-1) but did recognize a mutant gC-1 molecule. This conversion from a type 1 to a type 2 epitope was shown to be due to a single amino acid substitution in gC-1.     

A single amino acid change restores DNA cytosine methyltransferase activity in a cloned chlorella virus pseudogene.

The chlorella virus PBCV-1 contains an open reading frame, named P17-ORF4, which differs by eight amino acids from a DNA cytosine methyltransferase, M.CviJI, encoded by a different chlorella virus IL-3A. Whereas IL-3A expresses M.CviJI, which methylates the central cytosine in (A/G)GC(T/C/G) sequences, P17-ORF4 is non-functional. Gene fusions between P17-ORF4 and M.CviJI and site-directed point mutations revealed that changing Gln188 to Lys188 abolishes M.CviJI methyltransferase activity. Conversely, changing Lys188 in P17-ORF4 to Gln188 results in M.CviJI activity. The other altered seven amino acids do not appear to affect M.CviJI activity.     

Ngari virus is a Bunyamwera virus reassortant that can be associated with large outbreaks of hemorrhagic fever in Africa

Two isolates of a virus of the genus Orthobunyavirus (family Bunyaviridae) were obtained from hemorrhagic fever cases during a large disease outbreak in East Africa in 1997 and 1998. Sequence analysis of regions of the three genomic RNA segments of the virus (provisionally referred to as Garissa virus) suggested that it was a genetic reassortant virus with S and L segments derived from Bunyamwera virus but an M segment from an unidentified virus of the genus Orthobunyavirus. While high genetic diversity (52%) was revealed by analysis of virus M segment nucleotide sequences obtained from 21 members of the genus Orthobunyavirus, the Garissa and Ngari virus M segments were almost identical. Surprisingly, the Ngari virus L and S segments showed high sequence identity with those of Bunyamwera virus, showing that Garissa virus is an isolate of Ngari virus, which in turn is a Bunyamwera virus reassortant. Ngari virus should be considered when investigating hemorrhagic fever outbreaks throughout sub-Saharan Africa.     

Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein E(rns)

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.     

A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells.     

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.     

A single amino acid substitution in the transmembrane envelope glycoprotein of feline immunodeficiency virus alters cellular tropism.

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.