Minute Virus of Mice Small Nonstructural Protein NS2 Interacts and Colocalizes with the Smn Protein

Finally, we present evidence that NS-1 and NS-2 proteins are synthesized at apparently the same levels whether or not NS-3 is expressed.Densoviruses (DNVs) are autonomously replicating parvoviruses known to be highly pathogenic for two phyla of invertebrates: insects and crustacea (3, 13). Like their vertebrate counterparts, DNVs are small icosahedral nonenveloped viruses with a single-stranded linear DNA genome 4 to 6 kb in length (3,4,26). Based on the size, organization of coding sequences, and structure of extremities of their genome, DNVs are distributed in three genera within the subfamily Densovirinae (5). Members of the genus Densovirus, with the Junonia coenia DNV (JcDNV) as the prototype, have a 6-kb genome, a long (Ͼ500-nucleotide [nt]) inverted terminal repeat (ITR), and an ambisense organization. The four structural proteins VP1, VP2, VP3, and VP4 are nested in a single large open reading frame (ORF), ORF1, located in the 5Ј half of one strand, whereas in the 5Ј half of the complementary strand ORF2, ORF3, and ORF4 encode the three nonstructural (NS) proteins NS-1, NS-2, and NS-3, respectively (11). VP and NS genes are transcribed from the P9 and P93 promoters, respectively. The four overlapping capsid polypeptides are synthesized from an unspliced 2.5-kb mRNA by a "leaky-scanning" mechanism whereby ribosomes initiate translation at the first, second, third, or fourth/fifth in-frame AUG codon. Thus, the four VPs share the same C-terminal sequence and differ only in their N-terminal region. The frequency of translation initiation events at each AUG codon is very likely responsible for the different levels of expression of the four polypeptides, as reflected by the presence of VP1, VP2, VP3, and VP4 in an approximate 1:9:9:41 ratio in the virion (27). The three NS proteins are synthesized from two mRNAs, an unspliced 2.4-kb mRNA coding for NS-3 and a spliced 1.7-kb mRNA coding for NS-1 and NS-2 (unpublished data). Among other functions, these proteins are likely to be involved in viral DNA replication. Sequence analyses revealed that NS-1 proteins of DNVs share homologies with NS-1 proteins of vertebrate parvoviruses, including NTP-binding and helicase activity domains (1,3,11,26). Recent in vitro studies of JcDNV NS-1 biochemical properties have confirmed its involvement in viral DNA replication by showing that it binds to specific DNA sequences within the A-AЈ region of the terminal palindrome and, like its vertebrate counterparts, has multiple functions (including a strand-and site-specific nicking activity), and has an ATPdependent DNA helicase activity (10). In contrast, no clear function could be assigned so far to NS-2 and NS-3 proteins of DNVs. These proteins share no homology with NS proteins of vertebrate parvoviruses or with protein sequences from data banks. In the present study we attempted to elucidate the function of NS-3 in the life cycle of JcDNV by generating deletion mutants and analyzing their ability to undergo a replicative cycle. Our results demonstrate that NS-3 is absolutely

Section: Discussionmentioning

confidence: 99%

NS-3 Protein of the Junonia coenia Densovirus Is Essential for Viral DNA Replication in an Ld 652 Cell Line and Spodoptera littoralis Larvae

Abd-Alla¹,

Jousset²,

Li³

et al. 2004

“…However, nonphosphorylated NS2 can also be detected in the nuclei of infected cells (14). NS2 was shown to interact with at least two members of the 14-3-3 protein family (7) and with the survival motor neuron (SMN) protein (52,53). In addition, all three isoforms of NS2 shuttle from the nucleus to the cytoplasm by interacting with the nuclear export factor CRM1 (5,41).…”

mentioning

confidence: 99%

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

Weiss

Stroh-Dege

Rommelaere

et al. 2012

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...

“…NS1 is a nuclear phosphoprotein that is required for viral DNA replication (16) and is cytotoxic for cells at certain transformation stages (11,18). The smaller NS2 protein contains three isoforms (23 to 28 kDa) arising from alternate splicing (17) that can bind the cell cycle regulator 14-3-3 protein family (9) and the survival motor neuron (Smn) protein (59). NS2 shuttles from the nucleus to the cytoplasm by an interaction with the nuclear transport factor CRM1 (6,45) mediated by a conventional nuclear export sequence (NES) mapped between amino acids 81 and 103 of the viral protein (22,40,45).…”

mentioning

confidence: 99%

Enhanced Cytoplasmic Sequestration of the Nuclear Export Receptor CRM1 by NS2 Mutations Developed in the Host Regulates Parvovirus Fitness

López-Bueno

Valle

Gallego

et al. 2004

To investigate whether a DNA virus can evade passive immunotherapy with a polyclonal antiserum, we analyzed the protection of a neutralizing capsid antiserum against a lethal infection of the immunosuppressive strain of the parvovirus minute virus of mice (MVMi) in 42 immunodeficient mice over a period of 200 days. A few mice were effectively protected, but most developed a delayed lethal leukopenic syndrome during the treatment or weeks afterwards. Unexpectedly, viruses isolated from treated but also from control leukopenic mice showed no amino acid changes throughout the entire capsid coding region, although the viral populations were genetically heterogeneous, mainly in the second exon of the coding sequence of the NS2 nonstructural protein. The NS2 point amino acid changes (T88A, K96E, L103P, and L153 M) that were consistently selected in several mice clustered within the nuclear exportin CRM1 binding domain, in a reading frame that did not alter the overlapping NS1 coding region. These mutations endowed emerging viruses with an increased fitness that was demonstrable by their relative resistance to the neutralizing capsid antiserum in a postentry plaqueforming assay, the rapid overgrowth of a competing wild-type (wt) population in culture, and a larger yield of infectious particles. Mutant NS2 proteins interacted with a higher affinity and sequestered CRM1 in the perinuclear region of the cytoplasm more efficiently than the wt. Correspondingly this phenomenon, as well as the following timely ordered release of the NS1 nonstructural protein and the empty capsid from the nucleus to the cytoplasm, occurred markedly earlier in the infection cycle of the mutant viruses. We hypothesize that the enhanced cytoplasmic sequestration of CRM1 by the NS2 mutations selected in mice may trigger pleiotropic effects leading to an accelerated MVMi life cycle and thus to increased fitness. These results strengthen our earlier report on the rapid evolutionary capacity of this mammalian-specific DNA virus in vivo and indicate that the NS2-CRM1 interaction is an important determinant of parvovirus virulence that can be modulated in nature, hampering the effectiveness of passive antibody therapies in the long term.