Mutations in the Non-Structural Protein-Coding Sequence of Protoparvovirus H-1PV Enhance the Fitness of the Virus and Show Key Benefits Regarding the Transduction Efficiency of Derived Vectors

Abstract

Single nucleotide changes were introduced into the non-structural (NS) coding sequence of the H-1 parvovirus (PV) infectious molecular clone and the corresponding virus stocks produced, thereby generating H1-PM-I, H1-PM-II, H1-PM-III, and H1-DM. The effects of the mutations on viral fitness were analyzed. Because of the overlapping sequences of NS1 and NS2, the mutations affected either NS2 (H1-PM-II, -III) or both NS1 and NS2 proteins (H1-PM-I, H1-DM). Our results show key benefits of PM-I, PM-II, and DM mutations with regard to the fitness of the virus stocks produced. Indeed, these mutants displayed a higher production of infectious virus in different cell cultures and better spreading capacity than the wild-type virus. This correlated with a decreased particle-to-infectivity (P/I) ratio and stimulation of an early step(s) of the viral cycle prior to viral DNA replication, namely, cell binding and internalization. These mutations also enhance the transduction efficiency of H-1PV-based vectors. In contrast, the PM-III mutation, which affects NS2 at a position downstream of the sequence deleted in Del H-1PV, impaired virus replication and spreading. We hypothesize that the NS2 protein—modified in H1-PM-I, H1-PM-II, and H1-DM—may result in the stimulation of some maturation step(s) of the capsid and facilitate virus entry into subsequently infected cells.

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

“…Two mutant protoparvovirus H-1 strains, Del H-1PV [ 32 ] and DM H-1 [ 45 ], have been described as more efficient than wild-type H-1PV in progeny virus release and propagation in human cancer cell cultures. We have applied these strains to a panel of osteosarcoma cell lines characterized as semi-permissive to wild-type H-1PV infection, and have estimated their capacity to induce sustained cytotoxic effects.…”

H-1 Parvovirus as a Cancer-Killing Agent: Past, Present, and Future

“…NS1 is also the major effector of virus cytotoxicity (see below), and its expression is sufficient to trigger cell cycle arrest and apoptosis—similar to expression of the whole virus [34]. The role of H-1PV NS2 is less understood, but, based on studies on the closely related parvovirus MVM, it is thought to involve the modulation of viral DNA replication, viral mRNA translation, capsid assembly, and virus cytotoxicity [35].…”

“…In a subsequent study, Hashemi et al explored the effects of mutations within the H-1PV NS encoding region. By introducing into the H-1PV genome single nucleotide changes that have been shown to improve the fitness of the closely related lymphotropic strain of MVM, the authors generated H-1PV fitness variants with enhanced infectivity and transduction efficiency [35].…”

A Roadmap for the Success of Oncolytic Parvovirus-Based Anticancer Therapies

“…One such example is the H-1PV mutant H-1dr, which was isolated during routine plaque purification in an established simian virus 40-transformed newborn human kidney cell line (NB-E) and found to have a 114-nucleotide in-frame deletion in the NS gene causing internal deletions in the C-terminal regions of NS1 and NS2, as well as a 58-nucleotide duplication in the right palindromic sequence (68,94). This deletion mutant displayed improved fitness in human embryonic kidney NB-324K, pancreatic, and cervical carcinoma cell lines but lost the ability to infect rat fibroblasts (95,96).…”