Sequence analysis of the CXCR4 gene derived from cells surviving feline lentivirus infection.

The feline 3201-S cell line was established from cells that survived productive infection with feline immunodeficiency virus (FIV). We have recently shown that 3201-S cells are free of FIV DNA and are refractory to FIV reinfection. In addition, while the cells express CXCR4, a co-receptor for FIV infection, they are unresponsive to the CXCR4 ligand. In the present study, we show that 3201-S cells encode distinct mutations in the CXCR4 gene. It appears that 3201 cells are heterogeneous, consisting of phenotypically diverse mixed populations resulting from genetic mutations, suggesting that this defect can render the CXCR4 receptor expressed in 3201-S cells biologically dysfunctional.

Induction of feline immunodeficiency virus from a chronically infected feline T-lymphocyte cell line.

The infection of the feline T-lymphocyte cell line FeT-J with the feline immunodeficiency virus (FIV) Petaluma strain led to the establishment of nonvirus-producing cells. One clone (C15) obtained by limiting dilution was found to express FIV in response to chemical inducers of retroviruses. The chemical treatment of C15 cells led to not only FIV protein synthesis but also an augmentation of viral production. Examination of the C15 cell derivatives obtained by recloning revealed that 10-40% of treated cells constitutively expressed FIV antigens, whereas 100% with expressed FIV antigen in response to the inducer. Chemical induction resulted in more than a 100-fold increase in infectious viral production. The results suggest that a majority of FeT-J cells that are infected with FIV exist in a non-productive state. Establishing a cell line that can be non-productively infected by FIV may help determine the mechanisms of FIV latency.

Differential CXCR4 expression and function in subpopulations of the feline lymphoma cell line 3201 susceptible to feline immunodeficiency virus.

The infection of feline thymic lymphoma 3201 cells with a cell culture-adapted Petaluma strain of feline immunodeficiency virus (FIV) led to the establishment of survivor cells designated as 3201-S after a productive infection associated with extensive cell killing. 3201-S cells were free of FIV DNA, and were found to express CXCR4, a coreceptor for infection but not CD134, a primary receptor. When 3201-S cells were reinfected with FIV, viral DNA was transiently detectable for 5 days postinfection, indicating that 3201-S cells cannot support the FIV replicative cycle. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive 3201 cells, 3201-S cells did not show a flux of Ca(2+) in response to SDF-1alpha, implying that CXCR4 is not functionally active on 3201-S cells. These results suggest that 3201 cells can be heterogeneous in the phenotype of the CXCR4 expressed, and this heterogeneity may account for the differences in susceptibility to FIV. Determining the mechanism(s) within 3201-S cells that restrict FIV could result in therapeutic strategies against FIV infection.

Studies on the conditions required for structural and functional maturation of rabies virus glycoprotein (G) in G cDNA-transfected cells.

When the rabies virus G cDNA was expressed with the help of T7 RNA polymerase provided by a recombinant vaccinia virus (RVV-T7), functional G proteins were produced in terms of their ability to induce low pH-dependent syncytium formation and the formation of conformational epitopes, including the acid-sensitive epitope recognized by mAb #1-30-44. Such an ability and the 1-30-44 epitope formation, however, were not associated with the G gene products when G cDNA was expressed without the help of RVV-T7 using a tetracycline-regulated expression vector (pTet-G), although they were normally transported to the surface of established G protein-producing BHK-21 (G-BHK) cells. But, when the G-BHK cells were treated with 2.5 m M sodium butyrate (NaB) after the removal of tetracycline, we could observe not only a much increased frequency of G protein-producing cells, but also the greatly enhanced maturation of the protein. Another short acylate, sodium propionate (NaP), similarly induced increased G protein synthesis at a concentration of 2.5 m M as NaB; however, such proteins were mostly not endowed with the fusion activity nor the 1-30-44 epitope, while NaP at a higher concentration as 5.0 m M did induce similarly the increased production and enhanced maturation of G protein, including the 1-30-44 epitope formation. From these results, we conclude that functional maturation of G protein to acquire fusogenic activity is correlated with 1-30-44 epitope formation, and 2.5 m M NaB not only stimulates G protein production, but also provides such cellular conditions as are required for the structural and functional maturation of the protein.

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production.

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.

Mapping of the low pH-sensitive conformational epitope of rabies virus glycoprotein recognized by a monoclonal antibody #1-30-44.

Monoclonal antibody (mAb) #1-30-44 recognized an acid-sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP-Flury strain and the epitope-negative CVS strain as well as the mAb-resistant escape mutants, two distant sites that contained Lys-202 and Asn-336 were shown to be involved in the epitope formation. Lys-202 is located in the so-called neurotoxin-like sequence, while Asn-336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP-Flury strain, in which Gln-333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1-30-44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin-like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1-30-44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH-dependent function of G protein.

Further characterization of the rabies virus glycoproteins produced by virus-infected and G cDNA-transfected cells using a monoclonal antibody, #1-30-44, which recognizes an acid-sensitive epitope.

Expression of rabies virus glycoprotein (G) by G cDNA-transfected mammalian cells resulted in the production of only a fusion-negative form. Low pH-dependent fusion activity, however, was seen when the expression was done under control of the T7 promoter with the help of recombinant vaccinia virus (RVV-T7) that provided T7 RNA polymerase. Fusion-inactive G proteins were transported to the cell surface as being detected by a conformational epitope-specific monoclonal antibody (mAb; #1-46-12). The fusion-inactive G proteins were recognized by most of our 13 conformation-specific mAbs, except for one mAb, #1-30-44, that recognized the low pH-sensitive conformational epitope. When the G gene expression was done with the help of RVV-T7, although most G proteins remained in the epitope-negative form, a small fraction of G gene products were 1-30-44 epitope-positive, and cell fusion activity could be seen when cells were exposed to low pH conditions. From these results, we conclude that acquisition of low pH-dependent fusion activity is closely related to structural maturation of the G protein to form the low pH-sensitive 1-30-44 epitope. Such maturation seems to be dependent on certain rabies virus-induced cellular conditions or functions, which might also be provided in part by the vaccinia virus infection. We further assume that expression of G cDNA alone mostly results in the production of mis-folded and/or differently folded forms of G protein, and only a small fraction is correctly folded even under RVV-T7-mediated expression conditions.

Structural relationship between nucleocapsid-binding activity of the rabies virus phosphoprotein (P) and exposure of epitope 402-13 located at the C terminus.

The structural changes of the nominal phosphoprotein (P) of rabies virus using a monoclonal antibody, mAb #402-13, was investigated. This mAb recognized a linear epitope that was mapped roughly to a C-terminal region of the P protein, ranging from aa 256 to 297. The P gene products were detected by the mAb in immunoblot assays, the products of which were produced either in BHK-21 cells or in Escherichia coli cells. The mAb, however, detected very low levels of P gene products in immunoprecipitation assays. The mAb recognized the nucleocapsid (NC)-associated P proteins but recognized free P protein and free N-P complex produced in the infected cells much less efficiently. When the P proteins were released from the NC, however, they were no longer recognized by the mAb. Similar results were obtained from BHK-21 cells co-transfected with P and N cDNAs. Furthermore, studies with C-terminally truncated P protein mutants revealed that the NC-binding ability of the P protein was dependent on the presence of the C-terminal epitope region. From these results, it is thought that the 402-13 epitope region is concealed when the P protein is present in a free form or free N-P complex but is exposed when it is associated with the NC. The C-terminal epitope region seemed to be essential for the P protein to be associated with the NC but not for the formation of free N-P complexes with newly synthesized N protein.