John H. Elder

Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose

A simple, economical, and efficient procedure for analysis of proteins (Western blotting) and DNA (Southern blotting) transferred to nitrocellulose for reaction with antibodies or nucleic acid probes is described. The techniques utilize nonfat dry milk as a protein-nucleic acid source for blocking nonspecific reactions, as an incubation medium, and for subsequent washing to remove unreacted reagents. The incubation cocktail, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer), is superior to bovine serum albumin or gelatin for preventing nonspecific absorption in Western blot analyses and does not require the use of detergents or chaotropic agents to effect efficient reduction of background. BLOTTO, at the proper dilution in NaClue5f8Na citrate, is just as efficient in Southern blot analyses as more complicated cocktails typically used in the latter technique. We also found that BLOTTO works well for blocking, incubating, and washing ELISA plate assays relative to the normal BSA carrier, at a considerable savings to the laboratory.

Regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus

The long terminal repeat (LTR) of a retrovirus contains sequence elements that constitute a promoter for controlling viral gene expression in infected cells. We have examined regulation of LTR-directed gene expression in feline immunodeficiency virus (FIV), a T-lymphocytopathic lentivirus associated with a fatal AIDS-like disease in domestic cats. Two independent virus isolates, designated FIV-Petaluma and FIV-PPR, have been molecularly cloned and show greater than 85% sequence homology. Both clones (termed pF34 and pPPR) produce infectious virus after transfection of permissive feline cells. Basal promoter activity of the LTRs was measured in various cell lines in transient expression assays using plasmids containing the viral LTR linked to the bacterial chloramphenicol acetyltransferase gene. Both LTRs were strong promoters in several cell lines, although in some cell lines the pF34 LTR had four- to fivefold higher basal activity than the pPPR LTR. FIV LTR mutations affecting the first AP4 site, AP1 site, ATF site, or NF-kappa B site resulted in decreased basal activity of the FIV promoter. Mutational analysis also revealed a negative regulatory element. In cotransfection experiments, both pF34 proviral DNA and pPPR proviral DNA appeared to transactivate either the pF34 LTR or the pPPR LTR; however, levels of transactivation were very low. Cotransfection of both LTRs with FIV subgenomic clones containing various viral open reading frames resulted in low level or no transactivation. The LTRs of both FIV clones responded to cell activation signals in human T-lymphoid cells (Jurkat) treated with phytohemagglutinin and phorbol-12-myristate-13-acetate. Promoter function of both FIV LTRs was also enhanced in cells treated with either forskolin, an inducer of intracellular cyclic-AMP (c-AMP), or dibutyryl c-AMP. Analysis of site-specific mutants showed that a potential AP1 site in the U3 domain of the LTR was required for T-cell activation responses mediated by protein kinase C, whereas a putative ATF site was the target for c-AMP-induced responses mediated by protein kinase A. These studies revealed that cellular transcription factors play a significant role in regulation of FIV gene expression.

Protein structure of lymphocytic choriomeningitis virus: identification of the virus structural and cell associated polypeptides.

Abstract Lymphocytic choriomeningitis virus (LCMV) has been found to contain 3 major structural polypeptides. The largest of these was shown to be a nonglycosylated nucleoprotein (NP) of approximately 63,000 daltons which was found internally in the virion and was associated with a dense ribonucleoprotein complex. Two glycopeptides, termed GP-1 and GP-2, of approximately 54,000 and 35,000 daltons, respectively, were shown to be localized on the virion surface by proteolytic digestion of intact virus. Peptide maps of GP-1 and GP-2 indicated that they were independent polypeptides and that GP-2 was not a cleavage product of GP-1. Furthermore, the tryptic peptide map of NP was distinct from the two glycopeptides. Pulse-labeling studies of the intracellular synthesis of LCMV specific polypeptides in infected BHK-21 cells demonstrated synthesis of the nucleoprotein by 6 hr after infection at a multiplicity of infection of 10, corresponding to the beginning of log phase of viral replication. In cytosol extracts of LCMV-infected cells enhanced by immune precipitation an additional apparently nonstructural glycopeptide with a molecular weight of 74–75,000 was observed. This cell associated glycopeptide, designated GP-C, was readily detectable after 24–48 hr of infection, and appeared to accumulate with increasing time of incubation. While the relationship of GP-C to the virion glycopeptides GP-1 and GP-2 has not been determined, immune precipitation studies have shown that GP-C does not share antigenic determinants with NP. The probable relationships between the polypeptides of lymphocytic choriomeningitis virus both to the pathogenesis of virus induced immune diseases and to the structure of other arenaviruses have been discussed.

Biological, immunological, and biochemical evidence that HIX virus is a recombinant between moloney leukemia virus and a murine xenotropic C type virus

Abstract Various parameters of HIX virus were examined to determine its origin and its relationship to other murine C type oncornaviruses. Envelope properties of HIX virus grown in cells of several species were subjected to analyses of host range, interference, and neutralization. Cloned amphotropic HIX virus was adapted to grow in human RD cells. After 6 months in culture, the resulting virus (HIX-RD) could enter mouse cells but essentially lost the capacity of propagating in mouse cells. Interference patterns of HIX and HIX-RD were identical to each other and unrelated to murine ecotropic MuLV interference. MSV(HIX) or MSV(HIX-RD) could not penetrate HIX-, HIX-RD-, or MuX-preinfected cells. However, infection with HIX exhibited a unique one-way interference in that MSV(MuX) could penetrate and transform HIX-preinfected cells. Neutralization of HIX and HIX-RD with relatively type-specific anti-gp70 sera showed that they resembled Moloney (M)-MuLV most closely. Significant neutralization was observed also with anti-Rauscher gp70 or BALB-2 MuX gp70 sera. Both HIX derivatives were acutely susceptible to inactivation with normal mouse sera, a characteristic of xenotropic viruses. Competition radioimmunoassays were performed to determine the antigenic relationship of HIX to other MuLV types. The highly type-specific phosphorylated p12 and the relatively type-specific gag region p15 of HIX were found to be identical to M-MuLV and less related to other murine C-type oncornaviruses. The examination of HIX gp70 with type-specific anti-M-MuLV or anti-C57L MuX gp70 sera showed that it was clearly different from either virus. Tryptic peptide maps of the gag region-p15 and p30 of HIX were identical to corresponding maps of M-MuLV proteins. The gp70 of HIX was unique and different from known eco-, xeno-, and amphotropic murine C type oncornaviruses. Based on known migration patterns of characteristic trypsin- and chymotrypsin-derived peptides of various eco-, and xenotropic MuLVs, it was concluded that gp70 of HIX was related to both MuX and M-MuLV. Tryptic fingerprint maps also revealed several significant differences between parental HIX and its HIX-RD variant. Comparative hybridizations of assorted high-molecular-weight (HMW) virus RNAs with complementary DNA from HIX virus showed that, with unfractionated probes, no significant differences could be seen between HIX and M-MuLV. Based on the above, HIX virus appears to contain predominantly M-MuLV-specific information except for its envelope gene which has been presumably derived from a recombinational event involving corresponding M-MuLV and MuX nucleotide sequences.

Endoglycosidases from Flavobacterium meningosepticum application to biological problems

Publisher Summary This chapter describes the purification and properties of these useful enzymes as well as some of the biological problems to which they have been applied. The enzymes are useful in studying (1) the role of N-linked carbohydrate side chains in the stability of glycoproteins and protection from proteolytic attack, (2) the influence of N-linked glycans on immune surveillance of glycoproteins, which is particularly relevant to the immune response against enveloped viruses, (3) the role of oligosaccharide side chains in specific intercellular interactions, (4) the precursor-product relationships of glycoproteins and whether molecular polymorphisms are because of multiple gene products or differential glycosylation of a single polypeptide backbone, (5) the use of potential sites of glycosylation predicted from nucleotide sequencing, (6) the isolation and detailed structural characterization of N-linked oligosaccharides, and (7) the role of N-linked carbohydrates on the biological activities of glycoprotein enzymes and hormones.

Differential expression of two distinct xenotropic viruses in NZB mice.

Abstract Two distinct xenotropic viruses have been isolated from New Zealand Black (NZB) mice. One of these viruses (NZB-X1) is spontaneously produced by NZB and NZB hybrid mice. The second xenotropic virus (NZB-X2) is induced by treatment of NZB fibroblasts with IdU. NZB-X1 is distinct in that it possesses a unique surface glycoprotein (gp70) similar to gp70 found free in the serum of all mouse strains tested, but only associated with a virion in NZB mice. NZB-X2 has a gp70 similar to that of xenotropic viruses isolated from mouse strains other than NZB, including xenotropic viruses of NIH Swiss, AKR, NZW, C57BL 6 BALB c , and wild mice, although NZB-X2 gp70 shows more structural relatedness to NIH Swiss and wild mouse xenotropic gp70 than to the others. By backcrossing NZB to NFS Swiss mice, two independently segregating loci have been described which control high and low xenotropic virus expression. Analyses of virus isolates from mice possessing these segregated loci showed that the high expression locus is linked to expression of the spontaneously released NZB virus, NZB-X1. The presence of the low expression locus corresponds with the production of the second xenotropic virus released after IdU treatment, NZB-X2. Using unique structural markers for these two xenotropic viruses, we have examined the phenomena of differential expression of endogenous virus and viral antigens within the NZB mouse. We find, as reported previously, that the predominant gp70 of serum is similar to NZB-X1. However, gp70s expressed on thymus and spleen carry the determinants of NZB-X2. The expression of these gp70s was not a peculiarity of NZB mice, since others tested including C57BL 6 , NZW, and 129 mouse strains showed the same tissue segregation. The differential expression of these xenotropic virus gp70s noncoordinate with virus production points to differential expression of endogenous envelope genes along different differentiation pathways.

A simple and rapid procedure for the purification of plasmid DNA using reverse-phase C18 silica beads.

A simple and efficient procedure for the rapid isolation of plasmid DNA free of chromosomal DNA and with only minor contamination with RNA is described. The protocol is a modification of the boiling method described by Holmes and Quigley [(1981) Anal. Biochem. 114, 193-197.] and utilizes C18 reverse-phase silica beads for final concentration and purification of plasmid DNA. The entire procedure can be carried out in 1 day and does not require the use of phenol or cesium chloride gradients, which require considerable labor and may sometimes cause nicking and lower recoveries of supercoiled DNA. The plasmid DNA obtained by this method retains biological activity, is supercoiled, and is suitable for restriction and DNA sequence analysis.

Pathogenesis of the slow disease of the central nervous system associated with wild mouse virus. III. Role of input virus and MCF recombinants in disease

Inbred mice bearing the FV-1n marker when inoculated at birth with an ecotropic murine leukemia virus (WM 1504-E) obtained from wild mice develop a progressive central nervous system (CNS) disease manifested by hindlimb paralysis and incoordination that begins by 6 to 7 weeks of age. Studies of infected SWR/J mice at 3 days to 4 months of age indicated the following: (1) Expression of MuLV gp69/70 and p30 antigens in CNS rises beginning as early as 3 days after inoculation and increases with time. (2) Neuronal damage is evident by Day 7, and neuronal lesions develop in all mice by Day 14. (3) WM 1504-E virus can be isolated from CNS tissue by 48 hr after initiating infection. (4) Upon passage into susceptible newborn mice, the WM 1504-E isolates cause neuronal disease. (5) Dual-tropic mink cell focus forming (MCF) -like virus is found in splenic but not CNS tissues by 8 weeks after initiating infection. (6) MCF viruses that arise by env gene recombination between WM 1504-E and endogenous xenotropic viruses do not cause CNS disease upon inoculation into susceptible newborn mice. Similarly inoculated WM 1504-amphotropic virus (WM 1504-A) does not cause CNS disease (7). Results in SWR/J mice can be duplicated in C3H/St and C57Br/cdj mice. These observations define the wild mouse ecotropic virus as the sole infectious agent responsible and sufficient for the development of this neurologic disease. Evidently, the disease from this slow virus infection begins early in life shortly after introduction of the infectious agent, but becomes clinically apparent only as neuronal destruction accumulates over the lifetime of the host.

Structural analysis of the major envelope glycoprotein (gp70) of the amphotropic and ecotropic type C viruses of wild mice

Abstract Virus-associated and free serum gp70s from wild mice ( Mus musculus ) were isolated by immunoaffinity column chromatography and were analyzed by two-dimensional tryptic peptide mapping procedures. Our results show that the gp70s of wild mouse ecotropic and amphotropic MuLVs are structurally divergent. The gp70s of wild mouse ecotropic viruses are more similar in structure to the Gross-AKR MuLV subgroup than to the FMR subgroup. The gp70s of the amphotropic wild mouse viruses are highly conserved in structure and are closely related to those of one group of xenotropic MuLVs. Non-virion-associated free serum gp70 of wild mice is homologous to that of inbred laboratory mouse strains.

Structural analysis of Rauscher virus Gp70 using monoclonal antibodies: Sites of antigenicity and p15(E) linkage

Abstract A linear map of 19 monoclonal antibody-binding domains on Rauscher retroviral gp70 was generated using a technique we have designated PEC-MAP (partial enzymatic cleavage-monoclonal antibody precipitation). We used eight proteolytic enzyme preparations in limited digests to produce 39 gp70 fragments. Immune precipitation of these fragments by monoclonal antibodies from 51 cell lines allowed us to define 19 binding sites by virtue of overlapping fragments and differential precipitation patterns. The sites most accessible to proteolytic attack and antibody binding were then mapped by analyzing the apparent molecular weights of the various gp70 fragments. This analysis revealed three hyperreactive regions in gp70 located approximately within the first 2000 daltons of the amino end of gp70 as well as 18,000 and 38,000 daltons from the amino end of the M r 47,000 deglycosylated gp70 molecule. In addition, we used monoclonal antibodies directed against the disulfide-linked p15(E) molecule to localize its linkage site to Domain XVII, estimated to be between 34,000 and 38,000 daltons from the amino end of deglycosylated gp70. These data place certain constraints on the tertiary structure of gp70 and suggest a mechanism for the generation of leukemogenic MCF recombinants.