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...

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.