Inability of Kaplan radiation leukemia virus to replicate on mouse fibroblasts is conferred by its long terminal repeat.

The molecularly cloned infectious Kaplan radiation leukemia virus has previously been shown to be unable to replicate on mouse fibroblasts (E. Rassart, M. Shang, Y. Boie, and P. Jolicoeur, J. Virol. 58:96-106, 1986). To map the viral sequences responsible for this, we constructed chimeric viral DNA genomes in vitro with parental cloned infectious viral DNAs from the nonfibrotropic (F-) BL/VL3 V-13 radiation leukemia virus and the fibrotropic (F+) endogenous BALB/c or Moloney murine leukemia viruses (MuLV). Infectious chimeric MuLVs, recovered after transfection of Ti-6 lymphocytes with these recombinant DNAs, were tested for capacity to replicate on mouse fibroblasts in vitro. We found that chimeric MuLVs harboring the long terminal repeat (LTR) of a fibrotropic MuLV replicated well on mouse fibroblasts. Conversely, chimeric MuLVs harboring the LTR of a nonfibrotropic MuLV were restricted on mouse fibroblasts. These results indicate that the LTR of BL/VL3 radiation leukemia virus harbors the primary determinant responsible for its inability to replicate on mouse fibroblasts in vitro. Our results also show that the primary determinant allowing F+ MuLVs (endogenous BALB/c and Moloney MuLVs) to replicate on mouse fibroblasts in vitro resides within the LTR.     

Host cell specificity of minute virus of mice in the developing mouse embryo

Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.     

Spontaneous curing of a minute virus of mice carrier state by selection of cells with an intracellular block of viral replication.

We previously described a persistent infection established by the lymphotropic minute virus of mice in mouse L cells at the level of the cell population (D. Ron, P. Tattersall, and J. Tal, J. Virol. 52:63-69, 1984). This carrier state is maintained by a series of consecutive phenotypic changes which take place in both the cells and the virus and is cured spontaneously after 150 to 200 cell generations (D. Ron and J. Tal, J. Virol. 55:424-430, 1985). We show here that the cure was caused by the selection of virus-resistant cells in the culture. The resistance of these survivor cells to virus replication was due to an intracellular block. Infection of a spontaneously cured culture with the fibrotropic parental minute virus of mice resulted in a restrictive infection in which the viral replicative-form DNA was formed and amplified, but the synthesis of single-stranded progeny DNA was markedly reduced. The lymphotropic strain was blocked in these cells at an earlier stage, with little or no amplification of viral replicative-form DNA observed. These data indicate that the replication of minute virus of mice requires host-coded helper functions in at least two stages of its growth cycle.     

Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells

Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus.     

DNA sequence of the lymphotropic variant of minute virus of mice, MVM(i), and comparison with the DNA sequence of the fibrotropic prototype strain.

The sequence of molecular clones of the genome of MVM(i), a lymphotropic variant of minute virus of mice, was determined and compared with that of MVM(p), the fibrotropic prototype strain. At the nucleotide level there are 163 base changes: 129 transitions and 34 transversions. Most nucleotide changes are silent, with only 27 amino acids changes predicted, of which 22 are conservative. Notable differences between the MVM(i) and MVM(p) genomes which may account for the cell specificities of these viruses occur within the 3' nontranslated regions. The differences discussed include the absence of a 65-base-pair direct in MVM(i), the presence of only two polyadenylation sites in MVM(i) compared with four in MVM(p), and sequences that bear a resemblance to enhancer sequences. Also included in this paper is an important correction to the MVM(p) sequence (C.R. Astell, M. Thomson, M. Merchlinsky, and D. C. Ward, Nucleic Acids Res. 11:999-1018, 1983).     

Studies on emerging radiation leukemia virus variants in C57BL/Ka mice.

To analyze the emergence of radiation leukemia virus (RadLV) variants in primary X-ray-induced C57BL/Ka thymoma and to identify the virus responsible for the very high leukemogenic potential of passaged Kaplan strain BL/VL3 preparation, we cloned several primary and passaged ecotropic RadLV infectious genomes. By restriction analysis, we found that BL/VL3 cells harbor three related but different ecotropic RadLVs. Their restriction map differs significantly from those of primary RadLVs. Hybridization analysis also indicated that BL/VL3 and primary RadLVs differ in their p15E and long terminal repeat (LTR) regions. As compared with the LTR sequence of the putative parental endogenous ecotropic provirus, the LTR sequence of primary weakly leukemogenic RadLV has only one change, a C-rich sequence, generating a 6-base-pair direct repeat just in front of the promotor. The LTR of the primary nonleukemogenic RadLV only showed few base changes, mainly clustered in R and U5. The LTR from a moderately leukemogenic passaged BL/VL3 RadLV had conserved the C-rich sequence and acquired a 43-base-pair direct repeat in U3 and several other point mutations, small insertions, and deletions scattered in U3, R, and U5. All cloned primary RadLVs were fibrotropic, and some were weakly leukemogenic. All cloned BL/VL3 RadLVs were thymotropic and nonfibrotropic. The block of their replication was found to be after the synthesis of unintegrated linear and supercoiled viral DNA. Most of the BL/VL3 RadLVs were moderately leukemogenic, and one (V-13) was highly leukemogenic, being as virulent as the Moloney strain. We propose a model for the emergence of the RadLV variants and show that the virus responsible for the high leukemogenic potential of BL/VL3 preparation is a nondefective, ecotropic, lymphotropic, nonfibrotropic, unique retrovirus which most likely arose from a parental primary RadLV similar to those studied here.     

The Human Cytomegalovirus UL133-138 Gene Locus Attenuates the Lytic Viral Cycle in Fibroblasts

The genomes of HCMV clinical strains (e.g. FIX, TR, PH, etc) contain a 15 kb region that encodes 20 putative ORFs. The region, termed ULb’, is lost after serial passage of virus in human foreskin fibroblast (HFF) cell culture. Compared to clinical strains, laboratory strains replicate faster and to higher titers of infectious virus. We made recombinant viruses with 22, 14, or 7 ORFs deleted from the ULb’ region using FIX and TR as model clinical strains. We also introduced a stop codon into single ORFs between UL133 and UL138 to prevent protein expression. All deletions within ULb’ and all stop codon mutants within the UL133 to UL138 region increased to varying degrees, viral major immediate early RNA and protein, DNA, and cell-free infectious virus compared to the wild type viruses. The wild type viral proteins slowed down the viral replication process along with cell-free infectious virus release from human fibroblast cells.     

A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range.

We have investigated what protein sequences are necessary for glycoprotein incorporation into Rous sarcoma virus (RSV) virions by utilizing the hemagglutinin (HA) protein of influenza virus. Two chimeric HA genes were constructed. In the first the coding sequence for the signal peptide of the RSV env gene product was fused in frame to the entire HA structural gene, and in the second the hydrophobic anchor and cytoplasmic domain sequences of the HA gene were also replaced with those from the RSV env gene. Both chimeric genes, expressed from a simian virus 40 expression vector in CV-1 cells, yielded functional HA proteins that were transported to the cell surface and were able to bind to erythrocytes. When the genes were expressed in combination with the RSV gag-pol gene region in QT6 cells by using a vaccinia virus-T7 expression/complementation system, virions that efficiently incorporated either chimeric protein were assembled. This result indicated that the presence of the RSV env membrane anchor and cytoplasmic sequences did not facilitate HA glycoprotein incorporation into virions. The presence of the RSV env signal sequence allowed the chimeric HA genes to be substituted into the RSV-derived BH-RCAN.HiSV viral genome in place of the RSV env gene. Both chimeric genomes yielded infectious virus that could infect human and avian cells with equal efficiency. These experiments demonstrate that a foreign glycoprotein, efficiently incorporated into virions lacking a native glycoprotein, can confer a broadened host range on the virus. Moreover, because the HA of influenza virus requires the acidic pH of the endosome in order to be activated, these results imply that foreign proteins can modify the normal route of entry of this avian retrovirus.     

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins.

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.     

Mapping the viral sequences conferring leukemogenicity and disease specificity in Moloney and amphotropic murine leukemia viruses.

The Moloney murine leukemia virus (MuLV) is a highly leukemogenic virus. To map the leukemogenic potential of Moloney MuLV, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA from Moloney and amphotropic 4070-A MuLVs. Infectious chimeric MuLVs were recovered by microinjection of recombinant DNA into NIH/3T3 cells and tested for their leukemogenic potential by inoculation into NIH/Swiss newborn mice. Parental Moloney MuLV and amphotropic 4070-A MuLV induced thymic and nonthymic leukemia, respectively, when inoculated intrathymically. With chimeric MuLVs, we found that the primary determinant of leukemogenicity of Moloney and amphotropic MuLVs lies within the 1.5-kilobase-pair ClaI-PvuI long terminal repeat (LTR)-containing fragment. The presence of additional Moloney env-pol sequences with the Moloney LTR enhanced the leukemogenic potential of a chimeric MuLV significantly, indicating that these sequences were also involved in tumor development. Since parental viruses induced different forms of leukemia, we could also map the viral sequences conferring this disease specificity. We found that the 1.5-kilobase-pair ClaI-PvuI LTR-containing fragment of Moloney MuLV was necessary and sufficient for a chimeric MuLV to induce thymic leukemia. Similarly, the same LTR-containing fragment of amphotropic MuLV was necessary and sufficient for a chimeric MuLV to induce nonthymic leukemia. Therefore, our results suggest that specific sequences within this short LTR-containing fragment determine two important viral functions: the ability to transform cells in vivo (leukemic transformation) and the selection of a specific population of cells to be transformed (disease specificity).     

Evolution to pathogenicity of the parvovirus minute virus of mice in immunodeficient mice involves genetic heterogeneity at the capsid domain that determines tropism

Very little is known about the role that evolutionary dynamics plays in diseases caused by mammalian DNA viruses. To address this issue in a natural host model, we compared the pathogenesis and genetics of the attenuated fibrotropic and the virulent lymphohematotropic strains of the parvovirus minute virus of mice (MVM), and of two invasive fibrotropic MVM (MVMp) variants carrying the I362S or K368R change in the VP2 major capsid protein, in the infection of severe combined immunodeficient (SCID) mice. By 14 to 18 weeks after oronasal inoculation, the I362S and K368R viruses caused lethal leukopenia characterized by tissue damage and inclusion bodies in hemopoietic organs, a pattern of disease found by 7 weeks postinfection with the lymphohematotropic MVM (MVMi) strain. The MVMp populations emerging in leukopenic mice showed consensus sequence changes in the MVMi genotype at residues G321E and A551V of VP2 in the I362S virus infections or A551V and V575A changes in the K368R virus infections, as well as a high level of genetic heterogeneity within a capsid domain at the twofold depression where these residues lay. Amino acids forming this capsid domain are important MVM tropism determinants, as exemplified by the switch in MVMi host range toward mouse fibroblasts conferred by coordinated changes of some of these residues and by the essential character of glutamate at residue 321 for maintaining MVMi tropism toward primary hemopoietic precursors. The few viruses within the spectrum of mutants from mice that maintained the respective parental 321G and 575V residues were infectious in a plaque assay, whereas the viruses with the main consensus sequences exhibited low levels of fitness in culture. Consistent with this finding, a recombinant MVMp virus carrying the consensus sequence mutations arising in the K368R virus background in mice failed to initiate infection in cell lines of different tissue origins, even though it caused rapid-course lethal leukopenia in SCID mice. The parental consensus genotype prevailed during leukopenia development, but plaque-forming viruses with the reversion of the 575A residue to valine emerged in affected organs. The disease caused by the DNA virus in mice, therefore, involves the generation of heterogeneous viral populations that may cooperatively interact for the hemopoietic syndrome. The evolutionary changes delineate a sector of the surface of the capsid that determines tropism and that surrounds the sialic acid receptor binding domain.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell.

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity.     

Measles viruses possessing the polymerase protein genes of the Edmonston vaccine strain exhibit attenuated gene expression and growth in cultured cells and SLAM knock-in mice

Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.     

Physical mapping of the paralysis-inducing determinant of a wild mouse ecotropic neurotropic retrovirus.

We have recently shown that a molecularly cloned ecotropic retrovirus, initially isolated from the brain of a paralyzed wild mouse, retained the ability to induce hind limb paralysis when inoculated into susceptible mice (Jolicoeur et al., J. Virol. 45:1159-1163, 1983). To map the viral DNA sequences encoding the determinant of paralysis, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from this neurotropic murine leukemia virus (MuLV) and from nonneurotropic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered after microinjection of NIH 3T3 cells with these recombinant DNAs, were inoculated into newborn SIM.S and SWR/J mice to test the paralysis-inducing potential. We found that the 3.9-kilobase-pair SalI-ClaI fragment of the neurotropic MuLV comprising the 3' end of pol and all env sequences was sufficient to confer the paralysis-inducing potential to chimeric viruses. Therefore, this region of the neurotropic MuLV genome most likely harbors the primary determinant of paralysis.     

ZWA: Viral genome assembly and characterization hindrances from virus-host chimeric reads; a refining approach

Viral metagenomics, also known as virome studies, have yielded an unprecedented number of novel sequences, essential in recognizing and characterizing the etiological agent and the origin of emerging infectious diseases. Several tools and pipelines have been developed, to date, for the identification and assembly of viral genomes. Assembly pipelines often result in viral genomes contaminated with host genetic material, some of which are currently deposited into public databases. In the current report, we present a group of deposited sequences that encompass ribosomal RNA (rRNA) contamination. We highlight the detrimental role of chimeric next generation sequencing reads, between host rRNA sequences and viral sequences, in virus genome assembly and we present the hindrances these reads may pose to current methodologies. We have further developed a refining pipeline, the Zero Waste Algorithm (ZWA) that assists in the assembly of low abundance viral genomes. ZWA performs context-depended trimming of chimeric reads, precisely removing their rRNA moiety. These, otherwise discarded, reads were fed to the assembly pipeline and assisted in the construction of larger and cleaner contigs making a substantial impact on current assembly methodologies. ZWA pipeline may significantly enhance virus genome assembly from low abundance samples and virus metagenomics approaches in which a small number of reads determine genome quality and integrity.     

Herpesvirus sylvilagus infects both B and T lymphocytes in vivo.

Herpesvirus sylvilagus infection of cottontail rabbits (Sylvilagus floridanus) was studied as a model of herpesvirus-induced lymphoproliferative disorders. Leukocytosis, splenomegaly, proliferation of T cells and virus production by lymphocytes characterized this infectious mononucleosis-like disease. Approximately two copies of circular herpesvirus sylvilagus genomes per cell were detected in spleen cells at 2 weeks postinfection, and circular genomes could still be observed after 4 months. Circular viral genomes were found in both B and T lymphocytes. Small amounts of linear viral DNA (0.1 to 0.3 copies per cell) were also detected in both B and T cells. These results indicated that the virus did not replicate in the majority of lymphocytes in vivo. Herpesvirus sylvilagus infection in cottontail rabbits could be useful as a model for studying the complex virus-host relationships of lymphotropic herpesviruses and perhaps as an animal model for Epstein-Barr virus infection in humans.     

Variable region of betanodavirus RNA2 is sufficient to determine host specificity

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.