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

Abstract

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.

Replication of Minute Virus of Mice DNA Is Critically Dependent on Accumulated Levels of NS2

“…We found that the previously characterized NS2 mutation (14) which increases NS2-Crm1 interaction above wild-type levels could rescue ssDNA replication in fibroblasts in the MVMi background, in spite of the reduced levels of NS2 in these cells. NS2 mutants that generate wild-type levels of an NS2 protein that no longer interacts with Crm1 have multiple restrictions Marker lane contains double-stranded DNA fragments of (from bottom) 2.5, 5.0, and 7.5 kb.…”

“…Recently, Lopez-Bueno et al isolated a group of MVMi mutants that were selected for the ability to escape passive immunotherapy in immunosuppressed mice (14). Interestingly, a number of these viruses contained muta- tions in NS2 that increased the affinity of NS2 with Crm1.…”

“…Interestingly, a number of these viruses contained muta- tions in NS2 that increased the affinity of NS2 with Crm1. We reasoned that if the failure of MVMi to generate ssDNA following transfection of A9 cells is due to loss of a required NS2-Crm1 interaction, then perhaps an NS2 mutation which increases NS2-Crm1 interaction above wild-type levels [K96E/ L103P (14)] can rescue ssDNA replication in fibroblasts in the MVMi background, in spite of the reduced levels of NS2 in these cells. Introduction of this mutation into the infectious clone of MVMi restores its ability to replicate ssDNA following transfection of A9 cells (Fig.…”

“…Mutations were made using standard PCR techniques, as described previously (21), and were sequenced to ensure that they were as expected. The mutation which increased NS2 interaction with Crm1 was K96E/L103P, as described by Lopez-Bueno et al (14). The U2 snRNA mutations were made in the wild-type human U2 snRNA gene, obtained from Alan Weiner (29), by using standard techniques.…”

“…A reciprocal mutation, changing the A residue to G in the MVMp background, reduced ssDNA production following transfection into fibroblasts, showing that ssDNA production in the first round following transfection was correlated to accumulated levels of NS2. A mutation previously characterized by the Almendral lab to increase NS2-Crm1 interaction above wild-type levels (14) was found to rescue ssDNA replication in fibroblasts in the MVMi background, suggesting that a threshold level of this function was required for MVM replication in fibroblasts. The necessity of NS2 for replication in murine cells was further underscored by the observation that, although bearing a 3Ј splice site that performs poorly in fibroblasts, MVMi generated at least as much R2 and NS2 in murine lymphocytes as did MVMp in fibroblasts.…”