James C. Chan

Monoclonal and polyclonal antibody studies of VSV(hrMMTV) pseudotypes

Abstract By phenotypic mixing between the host range (hr) variant of mouse mammary tumor virus (MMTV) and the is-045 mutant of vesicular stomatitis virus (VSV), we were able to produce high titers of “complete” pseudotypes bearing the VSV genome and hrMMTV envelope. Four “strains” of pseudotypes designated VSV(C 3 H), VSV(GR), VSV(RIII), and VSV(C 3 Hf) were generated. To find out whether group-specific and type-specific neutralizing antigenic determinants (epitopes) were expressed on some of these pseudotypes, we utilized several monoclonal antibodies to MMTV-gp52 and sera from mammary tumor-bearing C 3 H/HeN mice in neutralization assays. Four monoclonal antibodies to MMTV-gp52, which were group specific in solid-phase MMTV binding assays, were found to neutralize the infectivity of VSV(C 3 H), VSV(GR), VSV(RIII), and VSV(C 3 Hf), indicating the expression of group-specific “neutralizing epitopes” on these pseudotypes. One monoclonal antibody to MMTV-gp52 that was C3Hf type specific in MMTV binding assays was found to neutralize only the infectivity of VSV(C 3 Hf) and not VSV(C 3 H), VSV(GR), or VSV(RIII). In addition, polyclonal antibodies were found in sera of mammary tumor-bearing C 3 H/HeN mice which neutralized specifically the infectivity of VSV(C 3 H). Such antibodies were not found in the sera of normal C 3 H/HeN mice or normal BALB/c mice. These results indicate that group-specific “neutralizing epitopes” were expressed on VSV(C 3 H), VSV(GR), VSV(RIII), and VSV(C 3 Hf). Furthermore, they suggest that type-specific “neutralizing epitopes” were expressed on VSV(C 3 H) and VSV(C 3 Hf) pseudotypes.

Sequence relationships between kirsten retrovirus genomes and the genomes of other murine retroviruses

RNA sequence relationships between the genomes of the Kirsten murine sarcoma virus (MSV-K) complex, the Kirsten murine leukemia virus (MuLV-K) complex, the Gross murine leukemia virus (MuLV-G), and the Moloney murine leukemia virus (MuLV-M) were investigated. Sedimentation analyses revealed the expected 30 and 34 S RNA subunits in the MSV-K complex and a previously undetected 30 S RNA subunit accompanying the 34 S RNA subunit in the MuLV-K complex. Nucleic acid hybridization data indicated that each Kirsten virus 30 S RNA subunit had about 40% sequence homology with the RNA genome of MuLV-G, although these sequences were only partially homologous between the two 30 S subunits. In contrast, the MuLV-K 34 S RNA subunit had 96% sequence homology with the MuLV-G genome, whereas the MSV-K 34 S RNA subunit displayed only 71% sequence homology with the MuLV-G genome. Similar relationships were indicated by oligonucleotide fingerprinting. The oligonucleotide data, taken with published sequence data on the MuLV-G and MuLV-M genomes, enabled us to construct partial sequence maps of the MuLV-K 34 S RNA subunit and the MSV-K 34 and 30 S RNA subunits. The sequence arrangements indicated that (1) the MuLV-K 34 S RNA subunit is a variant of the MuLV-G genome; (2) the MSV-K 34 S RNA subunit is a recombinant molecule, which maintains the length of its leukemia virus parent; and (3) the MSV-K 30 S RNA subunit may have been generated from the MuLV-K 34 S genome by a two-stage process, culminating in the retention of parental sequences only within the U5 and U3 noncoding segments and within several amino-terminal coding segments. Further examination of published retrovirus genome sequences revealed several strategically situated sets of potential recognition signals for transcription and translation and suggested a model for genetic recombination based on mRNA splicing signals and areas of limited sequence homology. This model may explain how foreign gene elements can be inserted into retrovirus genomes to generate either functional or defective recombinant retroviruses.

Molecular weight determination of glyoxalated RNA by sedimentation centrifugation

Abstract A rapid, reliable sedimentation centrifugation technique has been developed to measure the molecular weights of rather large glyoxalated RNAs. A distinctive feature of this method is that the glyoxalated RNAs can be analyzed in sucrose gradients containing no denaturant. This feature allowed us to compute the sedimentation coefficients of glyoxalated RNAs by a comparison with those of native, untreated RNA markers. These values then were used to obtain accurate molecular weight estimates by applying the linear log-log relation between the molecular weight of an RNA and its sedimentation coefficient.

Detection of VSV(MuLV) pseudotypes by an immunobiochemical technique

Abstract Vesicular stomatitis virus (murine leukemia virus) (VSV(MuLV)) pseudotypes containing a [ 3 H]uridine-labeled VSV RNA genome and MuLV envelope glycoproteins (gp70) were produced by phenotypic mixing of the two viruses. In order to better detect such pseudotypes, an immunobiochemical (IB) technique was developed. [ 3 H]Uridine-labeled virus progeny of the dual virus infection was immunoprecipitated by monospecific MuLV gp70 antibodies complexed with fixed Staphylococcus aureus . The immunoprecipitated 3 H-labeled genomic RNA was identified as that of VSV by its sedimentation coefficient, by the lack of polyadenylate, and by molecular hybridization with complementary VSV RNA. By the IB technique, approximately 11% of the progeny of the dual virus infection were found to be VSV(MuLV). By neutralization and other biological assays, however, only 0.1% of the progeny were found to be VSV(MuLV) pseudotypes. Apparently, the IB technique is capable of detecting VSV pseudotypes encapsidated with only a few molecules of MuLV gp70. The IB technique, therefore, offers a quantitative and molecular technique for the detection of VSV(MuLV) pseudotypes and can be modified to detect other viral pseudotypes when other assays are lacking. In spite of its sensitivity however, the IB technique did not detect the formation of MuLV (VSV) pseudovirions among the virus progeny of the dual virus infection. These results confirmed a similar observation made previously using immunoelectron microscopy.

Intracellular RNA complementary to the RNA genome of the Moloney--murine sarcoma virus complex.

Abstract Intracellular RNA complementary to genomic RNA of the Moloney-murine sarcoma virus complex was detected in virus-producing rat cells. Hybrids formed between this novel type of RNA and its homologous viral genomic RNA were very stable exhibiting a T m of 88°. Further characterization of the virus-specific complementary RNA revealed that it represented a minimal 74% of the viral RNA genome. Cross-hybridization data of hybrids formed between this viral complementary RNA and normal rat liver RNA or genomic RNA of several other mammalian retroviruses demonstrated its virus specificity and sequence relatedness. An examination of total cellular RNA from virus-producing cells for the number of intracellular virus-specific RNA copies disclosed 14 copies of viral complementary RNA per cell and 1272 copies of viral genomic RNA per cell.

Detection of vesicular stomatitis virus (murine leukemia virus) pseudotypes by immunoelectron microscopy.

Abstract Superinfection of a murine leukemia virus (MuLV)-producing rat bone tumor cell culture with the temperature-labile vesicular stomatitis virus (VSV) mutant tl-17 led to the formation of VSV(MuLV) pseudotype. This pseudotype was conveniently detected by immunoelectron microscopy (IEM) as bullet-shaped VSV particles reacting with anti-MuLV serum. In addition, VSV(MuLV) pseudotype formation was confirmed by results of thermoinactivation and neutralization tests. It is expected that the IEM described herein will find broader application to the demonstration of other pseudotypes that cannot be readily assayed by conventional infectivity assays.

Inhibition of the Release of Virion-Associated Murine Leukemia Virus Genomic RNA by Vesicular Stomatitis Virus

The genomic RNAs of murine leukemia virus (MuLV) and vesicular stomatitis virus (VSV) were distinguishable based on a difference in sedimentation coefficient, polyadenylic acid content, and nucleotide sequence homology. Applying these biochemical characteristics, we found that the release of virion-associated MuLV genomic RNA from virus-producing cells was inhibited by VSV as early as 2 h after superinfection. This finding offers an explanation to our previous inability to detect MuLV(VSV) pseudotype formation during mixed infection with VSV and MuLV.