Characterization of the cell type-specific determinant in the genome of minute virus of mice.

Two strains of minute virus of mice (MVM) show different host cell specificities. The prototype strain MVM(p) grows in fibroblasts, whereas the immunosuppressive variant MVM(i) grows in T lymphocytes. In this study, we have mapped on the viral genome a cell type-specific determinant: it is located between 69 and 85 map units in a region coding for the viral capsid proteins. The DNA of MVM(p) does not replicate in lymphocytes. MVM(i) cannot help MVM(p) grow in lymphocytes; thus the determinant acts in a cis fashion. We did not detect viral mRNA during a restrictive infection of lymphocytes with MVM(p). However, when the same cells were transfected with cloned DNA, both MVM(p) and MVM(i) DNAs were transcribed with the same efficiency from both promoters and the RNA was processed normally. Therefore, the specificity determinant is not a cell type-specific enhancer.     

The mismatched nucleotides in the 5'-terminal hairpin of minute virus of mice are required for efficient viral DNA replication.

The 5'-terminal sequence in the DNA of the parvovirus minute virus of mice (MVM) is a palindrome. It can form a hairpin, the stem of which is entirely base-paired except for three consecutive unpaired nucleotides which form a bubble. Since this structure is well conserved among different parvoviruses, we examined its importance for viral replication by generating MVM mutants with alterations in this region. A clone of MVMp DNA which contained the entire 3' end and more than half of the 5' palindrome was made. Although it lacked the sequence information to form a wild-type bubble, this DNA was infectious. On transfection into A9 fibroblasts, it gave rise to a virus (MVMs) which had a bubble in its 5' palindrome. The bubble consisted of four mismatched nucleotides in the same location as the unpaired nucleotides of the wild-type palindrome. Apparently, neighboring plasmid sequences were incorporated into the viral DNA, enabling formation of the mismatch. This observation suggested that a bubble is critical for growth of MVM but that its sequence is not. To find out whether MVM lacking a bubble in the 5' palindrome is viable, we made a second clone in which the plasmid sequences incorporated in MVMs were removed. Transfection of this DNA gave rise to a virus (MVMx) in which the nucleotides unpaired in the wild-type hairpin are now fully base-paired. Although MVMx can be propagated, it is defective in comparison with wild-type MVMp; it exhibited about a 50-fold-lower ratio of plaque-forming units to DNA content. In mixed infections, MVMp consistently outgrew the bubbleless MVMx. The rate of accumulation of DNA replication intermediates was lower for MVMx than for the wild-type virus. Quantitative analysis of the 5' termini of replicative form DNA suggested that the ability of MVMx to convert hairpin 5' termini to extended termini is impaired. In contrast, the virus with the altered bubble, MVMs, behaved like the wild-type MVMp in all the assays. We conclude that MVM lacking a bubble in its 5'-terminal DNA hairpin is less infectious than and has a selective disadvantage compared with wild-type MVM. The nucleotide sequence of the bubble is not critical. We provide evidence that the presence of a bubble is necessary for efficient viral DNA replication.     

The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.     

Two amino acid substitutions within the capsid are coordinately required for acquisition of fibrotropism by the lymphotropic strain of minute virus of mice.

Nucleotide changes at both codons 317 and 321 in the VP2 capsid gene of the immunosuppressive strain of the murine parvovirus minute virus of mice, MVM(i), are required to create a virus capable of growing in A9 fibroblasts. This double mutant virus, ILB1, has growth characteristics very similar to those of the prototype fibrotropic strain MVM(p) in both single- and multiple-round infections of fibroblasts and is about 100-fold better at infecting fibroblasts than MVM(i). When only one nucleotide position is changed, either in codon 317 (as in ILB2) or in codon 321 (as in ILB3), the resulting viruses are less than twice as efficient as their parent MVM(i) at infecting fibroblasts. In the restrictive infection of A9 cells by the single mutants and MVM(i), gene expression and DNA replication were markedly reduced compared with ILB1 infection of the same cells or compared with infections of permissive hybrid cells by each of the viruses. This suggests that restriction acts predominantly at an early step in the infection. Since the phenotypes of ILB2 and ILB3 are essentially indistinguishable in restrictive infections, it is most likely that the individual loci affect the same step in the viral life cycle. The dramatic increase in fibroblast infectivity shown by ILB1 indicates a synergistic interaction between these two amino acid residues in the same rate-limiting process in fibroblast infection.     

Use of an autonomous parvovirus vector for selective transfer of a foreign gene into transformed human cells of different tissue origins and its expression therein.

In this work, we report the transduction of a chloramphenicol acetyltransferase (CAT) reporter gene into a variety of normal and transformed human cells of various tissue origins. The vector used was MVM/P38cat, a recombinant of the prototype strain of the autonomous parvovirus minute virus of mice (MVMp). The CAT gene was inserted into the capsid-encoding region of the infectious molecular clone of MVMp genome, under the control of the MVM P38 promoter. When used to transfect permissive cells, the MVM/P38cat DNA was efficiently replicated and expressed the foreign CAT gene at high levels. By cotransfecting with a helper plasmid expressing the capsid proteins, it was possible to produce mixed virus stocks containing MVM/P38cat infectious particles and variable amounts of recombinant MVM. MVM/P38cat viral particles were successfully used to transfer the CAT gene and to express it in a variety of human cells. Both viral DNA replication and P38-driven CAT expression were achieved in fibroblasts, epithelial cells, T lymphocytes, and macrophages in a transformation-dependent way, but with an efficiency depending on the cell type. In transformed B lymphocytes, however, the vector was not replicated, nor did it express the CAT gene.     

Virions released from cells transfected with a molecular clone of human T-cell leukemia virus type I give rise to primary and secondary infections of T cells.

The ability of molecular clones of human T-cell leukemia virus type I (HTLV-I) to direct the synthesis of infectious virions has not previously been demonstrated. An HTLV-I provirus originating from an adult T-cell leukemia patient was cloned into a plasmid vector and is designated pCS-HTLV. This molecular clone was shown to direct the synthesis of viral mRNA and proteins in transiently transfected cells; in addition, virus structural proteins were released into the culture medium. Viral proteins were assembled into virions that sedimented at a buoyant density characteristic of retrovirus particles and whose morphology was verified by electron microscopy. Virions concentrated from transiently transfected cell supernatants were incubated with primary cord blood lymphocytes or with transformed T-cell lines to establish that these particles were infectious. Expression of spliced, viral mRNAs in the T-cell cultures after both primary and secondary infections with cell-free virus revealed that pCS-HTLV encodes an infectious provirus.     

Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus.

Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.     

Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production.

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.     

Expression of minute virus of mice structural proteins in murine cell lines transformed by bovine papillomavirus-minute virus of mice plasmid chimera.

Recombinant plasmids containing the genomes of both bovine papillomavirus type I and minute virus of mice (MVM) were constructed and used to transform mouse C127 cells. Transformed lines that express MVM gene products with high efficiency were isolated and characterized. These transformants synthesize large amounts of MVM structural polypeptides and spontaneously assemble them into empty virion particles that are released into the culture medium. These lines were, however, genetically unstable; they slowly generated subpopulations that failed to express MVM-specific proteins, and they possessed episomal DNA in which both MVM and bovine papillomavirus sequences were deleted or rearranged, or both. Clonal isolates of these transformants were also superinfectible by infectious MVM virus. Therefore, in spite of their instability, they should be useful host cell lines for transcomplementing mutations introduced into the MVM genome and for growing defective viruses as virions.     

Infectious virus replication in papillomas induced by molecularly cloned cottontail rabbit papillomavirus DNA.

The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication.     

The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner.

Seven mutations which affect only the small nonstructural protein NS2 were introduced into the infectious clone of the autonomous parvovirus, minute virus of mice (MVM). The majority of these mutants were severely defective for replication following transfection of normal host murine A9 fibroblasts; however, all were found to replicate more efficiently and produce infectious virus in certain other cell types, including human NB324K. The isolation of viral stocks from NB324K cells permitted a more detailed analysis of the mutant defect on A9 cells. NS2 mutant NS2-2018 was shown to be approximately 10-fold deficient for viral monomer replicative-form DNA production within a single-burst cycle in infected A9 cells and produced a reduced amount of progeny single strand. Mutant NS2-2018 generated wild-type levels of monomer replicative-form DNA on NB324K cells but made reduced levels of progeny single strand and small plaques on these cells. The accumulation of NS1 is reduced late in NS2-2018 infection of A9 cells, but NS1 accumulates to wild-type levels late in NB324K cell infections. NS1 nuclear localization is not dependent on NS2 in A9 or NB324K cells. These results indicate that NS2 participates in MVM DNA replication and is required for efficient viral growth. The requirement for NS2 during MVM replication is also host cell specific. This requirement is significantly more pronounced in the normal host murine A9 cells than in certain other cell types, including NB324K.     

Nuclear export of the nonenveloped parvovirus virion is directed by an unordered protein signal exposed on the capsid surface

It is uncertain whether nonenveloped karyophilic virus particles may actively traffic from the nucleus outward. The unordered amino-terminal domain of the VP2 major structural protein (2Nt) of the icosahedral parvovirus minute virus of mice (MVM) is internal in empty capsids, but it is exposed outside of the shell through the fivefold axis of symmetry in virions with an encapsidated single-stranded DNA genome, as well as in empty capsids subjected to a heat-induced structural transition. In productive infections of transformed and normal fibroblasts, mature MVM virions were found to efficiently exit from the nucleus prior to cell lysis, in contrast to the extended nuclear accumulation of empty capsids. Newly formed mutant viruses lacking the three phosphorylated serine residues of 2Nt were hampered in their exit from the human transformed NB324K nucleus, in correspondence with the capacity of 2Nt to drive microinjected phosphorylated heated capsids out of the nucleus. However, in normal mouse A9 fibroblasts, in which the MVM capsid was phosphorylated at similar sites but with a much lower rate, the nuclear exit of virions and microinjected capsids harboring exposed 2Nt required the infection process and was highly sensitive to inhibition of the exportin CRM1 in the absence of a demonstrable interaction. Thus, the MVM virion exits the nucleus by accessing nonconventional export pathways relying on cell physiology that can be intensified by infection but in which the exposure of 2Nt remains essential for transport. The flexible 2Nt nuclear transport signal may illustrate a common structural solution used by nonenveloped spherical viruses to propagate in undamaged host tissues.     

Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.

We constructed an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified.