Growth of the parvovirus minute virus of mice MVMp3 in EL4 lymphocytes is restricted after cell entry and before viral DNA amplification: cell-specific differences in virus uncoating in vitro

Abstract

Two murine parvoviruses with genomic sequences differing only in 33 nucleotides (8 amino acids) in the region coding for the capsid proteins show different host cell specificities: MVMi grows in EL4 T lymphocytes and MVMp3 grows in A9 fibroblasts. In this study we compared the courses of infections with these two viruses in EL4 cells in order to investigate at which step(s) the infection process of MVMp3 is interrupted. The two viruses bound equally well to EL4 cells, and similar amounts of MVMi and MVMp3 input virion DNA appeared in the nuclear fractions of EL4 cells 1 h after infection. However, double-stranded replicative-form (RF) DNA of the two viruses appeared at different times, at 10 h postinfection with MVMi and at 24 h postinfection with MVMp3. The amount of MVMp3 RF DNA detected at 24 h was very small because it was produced only in a tiny subset of the population of EL4 cells that proved to be permissive for MVMp3. Replication of doublestranded viral DNA in EL4 cells was measured after transfection of purified RF DNA, cloned viral DNA, and cloned viral DNA with a mutation preventing synthesis of the capsid proteins. In each of these cases, DNA replication was comparable for MVMi and MVMp3. Production of virus particles also appeared to be similar after transfection of the two types of RF DNA into EL4 cells. Conversion of incoming 32 P-labeled singlestranded MVM DNA to 32 P-labeled double-stranded RF DNA was detected only after RF DNA amplification, indicating that few molecules serve as templates for viral DNA amplification. We showed that extracts of EL4 cells contain a factor which can destabilize MVMi virions but not MVMp3 by testing the sensitivity of viral DNA to DNase and by CsCl gradient analyses of viral particles. We therefore conclude that the MVMp3 life cycle is arrested after the transport of virions to the nucleus and prior to the replication of RF DNA, most likely at the stage of viral decapsidation.

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

“…This similarity in glycan profile thus fails to explain the differences in tropism for these cells between these MVM viruses based on differential cell surface glycan receptor recognition. These observations support previous claims that the determinant of differential cell and tissue tropism for MVMp and MVMi is post cell entry [26,28,36,58,59]. These previous reports identified amino acids at and surrounding the depression at icosahedral 2-fold axes of the capsid, which differ between the viruses, as playing a crucial role in dictating these differences.…”

“…The viruses are reciprocally restricted for growth in each other's cell type. Cell binding assays show that both strains bind to and enter the restricted cell, but there is a block prior to replicative form DNA replication likely due to a block in uncoating which is dictated by the VP sequence [26][27][28]. Both viruses are however oncotropic and display oncolytic activity in vitro, and are both able to infect the transformed human cell line NB324K (SV40 transformed human newborn kidney fibroblast cells), suggesting that the restricting environment is eliminated in tumor cells.…”

Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery

“…For example, the parvovirus MVMi infects lymphocytes, but not fibroblasts, and its lack of replication in fibroblasts is apparently due to the inability of the capsid to uncoat in these cells. Also, the addition of neutralizing antibodies to poliovirus, influenza, vaccinia, or reovirus can inhibit uncoating and thereby prevent infection (Previsani et al, 1997;Rigg et al, 1989;Rodriguez et al, 1985;Virgin et al, 1994). Nature has realized that uncoating is a vital step in viral gene delivery and expression, and this efficient unpackaging is therefore one more lesson that synthetic vectors should learn from viruses.…”

Construction of Pathogenic Molecular Clones of Aleutian Mink Disease Parvovirus that Replicate Bothin Vivoandin Vitro

“…Perhaps the capsid proteins of the nonviable plasmids are incapable of assembling into particles, a hypothesis that is testable using expression in recombinant baculoviruses (Christensen et al, 1993;Wu et al, 1994). Alternatively, the defect might be in capsid protein transport, receptor recognition, or genome encapsidation functions (Ball-Goodrich and Previsani et al, 1997;Strassheim et al, 1994;Truyen and Parrish, 1992). In addition, it is conceivable that virus might be produced after the initial round of transfection but be incapable of initiating subsequent rounds of infection (Gardiner and Tattersall, 1988;Tattersall and Bratton, 1983).…”

“…Capsid proteins of a variety of nonenveloped viruses, including parvoviruses, influence tropism, host range, and pathogenicity by diverse mechanisms. In the MVM and the CPV/FPV systems, the determinants for host range and tropism are encoded within VP2 and map to the surface of the capsid (Ball-Goodrich and Chang et al, 1992;Gardiner and Tattersall, 1988;Parker and Parrish, 1998;Strassheim et al, 1994;Truyen and Parrish, 1992); however, the effect is exerted at an intracellular step after cell entry, before viral DNA amplification (Ball-Goodrich and Previsani et al, 1997). Furthermore, in the case of CPV/FPV, the in vivo and in vitro host range determinants are distinct, yet both are controlled by the capsid proteins.…”