An In-Frame Deletion in the NS Protein-Coding Sequence of Parvovirus H-1PV Efficiently Stimulates Export and Infectivity of Progeny Virions

Abstract

An in-frame, 114-nucleotide-long deletion that affects the NS-coding sequence was created in the infectious molecular clone of the standard parvovirus H-1PV, thereby generating Del H-1PV. The plasmid was transfected and further propagated in permissive human cell lines in order to analyze the effects of the deletion on virus fitness. Our results show key benefits of this deletion, as Del H-1PV proved to exhibit (i) higher infectivity (lower particle-to-infectivity ratio) in vitro and (ii) enhanced tumor growth suppression in vivo compared to wild-type H-1PV. This increased infectivity correlated with an accelerated egress of Del H-1PV progeny virions in producer cells and with an overall stimulation of the viral life cycle in subsequently infected cells. Indeed, virus adsorption and internalization were significantly improved with Del H-1PV, which may account for the earlier appearance of viral DNA replicative forms that was observed with Del H-1PV than wild-type H-1PV. We hypothesize that the internal deletion within the NS2 and/or NS1 protein expressed by Del H-1PV results in the stimulation of some step(s) of the viral life cycle, in particular, a maturation step(s), leading to more efficient nuclear export of infectious viral particles and increased fitness of the virus produced. R odent parvoviruses (PVs), including the rat parvovirus H-1PV, belong to the genus Parvovirus within the subfamily Parvovirinae, whose members are endogenous to vertebrates. These autonomous parvoviruses are small, nonenveloped viruses and display an icosahedral capsid in which a single-stranded DNA genome of about 5,000 nucleotides is packaged (17). The parvoviral genome mainly consists of two large overlapping open reading frames under the control of two promoters (11,17). The early promoter, P4, drives the expression of the viral nonstructural proteins NS1 and NS2 (18, 48), while the late promoter, P38, controls the expression of the viral capsid proteins VP1 and VP2 and of the nonstructural protein SAT (48,54). NS1 is a multifunctional, mainly nuclear, phosphorylated protein of about 83 kDa that is required for the replication of the viral genome, in particular, due to its helicase, endonuclease, ATPase, and site-specific DNAbinding activities (15,30). Besides its function in viral DNA replication, NS1 transactivates the P38 promoter (10,20,30), plays a critical role in the vesicular egress of progeny virions (1, 38), and is the main determinant of parvoviral cytotoxicity (30, 39). Like NS1, the small NS2 proteins (about 25 kDa) are essential for various steps of the parvovirus life cycle, but unlike NS1, they do not appear to display any enzymatic activity. The NS2 proteins of the parvovirus minute virus of mice, prototype strain (MVMp), consist of three isoforms that are generated by alternative splicing and differ in their carboxyl termini (14). Although NS2 has been reported to play a role during viral DNA replication (9, 15, 36), translation of viral mRNA (28, 29, 37), capsid assembly (12), and parvoviral cytotoxicity (6,8...

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

“…It has been hypothesized that the greater egress of infective viral progeny viruses and the higher infectivity observed in vitro with these mutants may lead to higher antineoplastic efficacy in vivo. This has been confirmed for Del H-1PV in pancreatic cancer and cervix carcinoma xenograft models [ 30 ].…”

“…Contamination of virus stocks with endotoxins was below 2.5 U/mL. The Del H-1PV mutant was produced as previously described [ 30 ].…”

“…In recent years, H-1PV fitness mutants have been isolated and tested for antineoplastic efficacy [ 30 ]. It has been hypothesized that the greater egress of infective viral progeny viruses and the higher infectivity observed in vitro with these mutants may lead to higher antineoplastic efficacy in vivo.…”

Genome Sequence of Tumor Virus X, a Member of the Genus Protoparvovirus in the Family Parvoviridae

“…TVX has an in-frame, 111 nucleotide deletion within the coding sequence of NS1 and the second exon of NS2, deleting amino acids 587 to 624 of NS1, and 96 to 132 of NS2. This deletion bears striking resemblance to that of an H-1 variant named H-1 dr (8, 9) but is one codon shorter at its 3′ end. TVX also harbors a 24 nucleotide insertion, encoding 8 in-frame amino acids, in the VP1-specific region of the coat protein, and a perfect 93 nucleotide repeat that spans the C-terminus of the capsid gene.…”

Standardized large-scale H-1PV production process with efficient quality and quantity monitoring

“…Current research in our lab aims at developing a portfolio of modified oncolytic protoparvoviruses, vector derivatives, and protoparvovirus combination treatments with enhanced anticancer action (Li et al, 2013;Weiss et al, 2012). These developments should yield proofs of concept to be put to the clinical test.…”