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PubMed1983

The high leukemogenic potential of Gross passage A murine leukemia virus maps in the region of the genome corresponding to the long terminal repeat and to the 3' end of env.

The Gross passage A murine leukemia virus (MuLV) is a highly leukemogenic, ecotropic fibrotropic retrovirus. Its genome is similar to that of other nonleukemogenic ecotropic fibrotropic MuLVs but differs at the 3' end and in the long terminal repeat. To determine whether these modifications were related to its leukemogenic potential, we constructed a viral DNA recombinant in vitro with cloned infectious DNA from this highly leukemogenic Gross passage A MuLV and from a weakly leukemogenic endogenous BALB/c B-tropic MuLV. Infectious viruses, recovered after microinjection of murine cells with recombinant DNA, were injected into newborn mice. We show here that the Gross passage A 1.35-kilobase-pair KpnI fragment (harboring part of gp70, all of p15E, and the long terminal repeat) is sufficient to confer a high leukemogenic potential to this recombinant.

AKR murine leukemia virus↗

PubMed1983

Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses.

The murine leukemia viruses (MuLVs) have different host ranges and were originally designated N-tropic and B-tropic if they replicated preferentially in vitro on NIH and BALB/c fibroblasts, respectively. It was later found that N-tropic MuLVs were in fact restricted in BALB/c cells, that B-tropic MuLVs were restricted in NIH cells, and that both viruses were restricted in (BALB X NIH) F1 cells. A single gene, Fv-1, with two alleles, Fv-1b and Fv-1n, determines this dominant restriction. A virus-encoded protein seems to carry the viral host range determinant which is recognized by the Fv-1 gene product. To map the viral DNA sequences encoding this determinant, we constructed viral DNA recombinants in vitro between the cloned infectious viral DNA genomes from BALB/c N-tropic and B-tropic MuLVs. Infectious recombinant MuLVs were recovered by microinjecting these recombinant DNAs into murine Fv-1- SC-1 cells and were subsequently tested in vitro for their host ranges (N- or B-tropic). We found that a short 302-base pair 5'-end fragment was necessary and sufficient to confer a specific host range to a recombinant. Our sequencing data revealed that this fragment codes for amino acid sequences in gag p30. They also showed that only two consecutive amino acid differences, Gln-ArgN- and Thr-GluB-, in p30 are responsible for the N- and B-tropic host ranges of the BALB/c MuLVs, respectively. Therefore, it appears that the Fv-1b and Fv-1n gene products can discriminate between these two p30 amino acid sequences.

Amino Acid Sequence↗

PubMed1985

Envelope gene and long terminal repeat determine the different biological properties of Rauscher, Friend, and Moloney mink cell focus-inducing viruses.

The nucleotide sequence of the envelope (env) gene and the long terminal repeat (LTR) of an infectious clone of Rauscher mink cell focus-inducing (R-MCF) virus has been determined and compared with the published env gene and LTR sequences of Friend (F)- and Moloney (M)-MCF viruses. The sequence shows that R-MCF virus, like other MCF viruses, is a recombinant virus. Its env gene contains sequences which were acquired from an env gene in the mouse genome and which confer on the MCF virus its dualtropic host range. Unlike F-MCF and M-MCF viruses, R-MCF virus will not replicate in NIH 3T3 cells. The deduced amino acid sequence for the gp70 of R-MCF differs from that of F- and M-MCF viruses by 15 amino acids between residues 49 and 138 of gp70. These differences in amino acid sequences may be responsible for the inability of R-MCF virus to replicate in NIH 3T3 cells. The host range of two hybrid viruses constructed in vitro is consistent with this hypothesis. R-MCF virus and Friend murine leukemia virus (F-MLV) show 98% identity in their env gene 3' from the acquired env sequences. This contrasts with 82% identity between the env gene of R-MCF virus and M-MLV. The LTR of R-MCF shows 98% identity with the LTR of F-MCF as compared to 88% identity with the LTR of M-MCF. This striking similarity between the sequences of R-MCF, F-MCF, and F-MLV is surprising since the Rauscher virus and the Friend virus are thought to have originated independently. The high degree of similarity suggests that Rauscher and Friend viruses have a common origin. In contrast to M-MLV, which induces predominantly a lymphoid disease, R- and F-MCF viruses induce an erythroproliferative disease in NIH Swiss mice. A hybrid R-MCF virus with a genome derived primarily from R-MCF virus and a 3' end including the U3 region derived from M-MLV induces a lymphoid disease instead of an erythroid disease. This result indicates that it is the U3 region which determines the tissue specificity of the MCF virus-induced disease. It is suggested that the putative viral enhancers in the U3 region play two roles in the process of leukemogenesis: in the Friend and Rauscher disease, the viral enhancers act by increasing the transcription of the MCF env gene; in the thymic lymphoma, the enhancers activate mainly the expression of cellular genes.

Amino Acid Sequence↗

PubMed1989

Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase.

Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.

Amino Acid Sequence↗

PubMed1990

A specific base transition occurs on replicating hepatitis delta virus RNA.

Three independent lines of evidence showed that when an infectious clone of hepatitis delta virus of known sequence was used to initiate genome replication, up to 41% of the genomes were specifically mutated in the amber termination codon (UAG to UGG) for the open reading frame of the delta antigen, thereby increasing the length of the predicted protein from 195 to 214 amino acids. This change was detected only on molecules that participated in RNA-directed RNA synthesis.

Amino Acid Sequence↗

PubMed1991

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome.

Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.

Animals↗

PubMed1991

Parvovirus NS1 stimulates P4 expression by interaction with the terminal repeats and through DNA amplification.

Parvovirus protein NS1 is required for replication of viral DNA and plays a role in the regulation of viral gene expression. NS1 trans-activates the P38 promoter for capsid protein synthesis and has variable effects on other promoters. In this study, we examined the effects of NS1 on the regulation of its own promoter, P4. A number of plasmid constructions were made with the P4 promoter fused to reporter genes. The effects of NS1 on expression from the P4 promoter differed depending on the construction. Plasmids containing viral sequences which could not replicate showed a decrease in P4 expression on cotransfection with the NS1 gene. However, plasmids having replication-proficient viral sequences showed a three- to fivefold increase in P4 expression dependent on NS1. The effect on NS1 on P4 transcription was also evaluated at the steady-state RNA level. An infectious clone of the LuIII viral genome was modified to an NS1-NS2 null mutant (pLu272) that is competent for viral DNA replication by introducing a frameshift mutation at codon 5 of the NS1 open reading frame. The P4 transcripts of pLu272 are four nucleotides longer than the wild type and can therefore be resolved from the wild type by primer extension analysis. pLu272 allows comparison of the constitutive level of steady-state RNA produced by the pLu272 P4 promoter in the absence or presence of a template replication dependent on NS1 supplied in trans. NS1 increased P4 transcripts about six- to eightfold. Expression of P4 transcripts from clones that could not amplify depended on the presence of an intact inverted terminal repeat sequence at the left end. A clone with an intact viral left end and a defective viral right end gave an NS1-dependent threefold increase in P4 expression. Destruction of terminal hairpins at both ends resulted in no significant increase in P4 expression in the presence of NS1. Thus, the positive effect of NS1 on the steady-state levels of P4 transcripts depends on the amplification of gene copy number and the integrity of the terminal repeats.

Base Sequence↗

PubMed1992

Processing of avian retroviral gag polyprotein precursors is blocked by a mutation at the NC-PR cleavage site.

The avian sarcoma and leukosis viruses (ASLV) encode a protease (PR) at the C terminus of gag which in vivo catalyzes the processing of both gag and gag-pol precursors. The studies reported here were undertaken to determine whether PR is able to cleave these polyproteins while it is still part of the gag precursor or whether the release of its N terminus to form free PR is necessary for full proteolytic activity. To address this question, we created a mutation that disrupts the PR cleavage site between the NC and PR coding regions of the gag gene. This mutation was introduced into a eukaryotic vector that expresses only the gag precursor and into an otherwise infectious clone of ASLV that carries the neo gene as a selectable marker. These constructs were expressed in monkey COS cells or in quail QT35 cells, respectively. Processing was impaired in both systems. Mutant particles were formed, but they contained no mature processed gag proteins. We observed only the uncleaved gag precursor polypeptide Pr76 in one case or Pr76 and a cleaved product of about 60 kDa in the other. Processing of the mutant gag precursor could be complemented in trans by from a wild-type construct, suggesting that the mutation did not induce gross structural alterations in its precursor. Our results suggest that the PR first must be released from its precursor before it can attack other sites in the gag and gag-pol polyproteins and that cleavage at the NC-PR boundary is a prerequisite for the initiation of the PR-directed processing.

Amino Acid Sequence↗

PubMed1992

The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.

Animals↗

PubMed1993

NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells.

Detailed analysis of five NS2 mutants of the autonomous parvovirus minute virus of mice (MVMp) has revealed the following. At low multiplicities of infection, NS2 mutants killed NB324K cells as well as wild-type (wt) MVM did and grew to high titers, while in contrast they grew poorly and did not readily kill murine A9 cells. Following CaPO4 transfection of murine fibroblasts, NS2 mutant infectious clones generated approximately 10-fold less monomer replicative-form DNA than wt and no detectable progeny single-stranded DNA. On nonmurine semipermissive NB324K cells, however, these mutant plasmid clones generated near wt levels of all replicative DNA forms. After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent. Both total and cytoplasmic NS2 mutant RNA was decreased, but the amount of total viral mRNA generated, relative to accumulated viral DNA in the same experiments, was similar to that seen in wt infection. The accumulation of virus-generated proteins was also decreased in NS2 mutant infection; however, the magnitude of this decrease, compared with that of wt infections, was significantly greater than the concomitant decrease in mutant-generated levels of accumulated cytoplasmic RNA, and this effect was most dramatic for VP2. There was no such disparity between the relative accumulation of mutant-generated RNA and protein in cells permissive for the growth of these mutants. These results suggest that translation of MVM viral RNA is specifically reduced in NS2 mutant infection of restrictive cells. Because the affected viral proteins are required for the efficient production of viral replicative DNA forms, these results reveal a fundamental, although perhaps not the only, role for NS2 in parvovirus infection.

Animals↗

PubMed1993

The major capsid protein VP2 of minute virus of mice (MVM) can form particles which bind to the 3'-terminal hairpin of MVM replicative-form DNA and package single-stranded viral progeny DNA.

The capsids of minute virus of mice (MVM) consist of two closely related proteins, VP1 and VP2. We inactivated the VP1 gene in an infectious clone of MVM DNA by frameshift mutation. After transfection of mutated DNA, capsids consisting of VP2 only were made. They can package negative-strand DNA, and they specifically bind MVM 3'-terminal hairpin DNA.

Animals↗

PubMed1995

HPRS-103 (exogenous avian leukosis virus, subgroup J) has an env gene related to those of endogenous elements EAV-0 and E51 and an E element found previously only in sarcoma viruses.

The avian leukosis and sarcoma virus (ALSV) group comprises eight subgroups based on envelope properties. HPRS-103, an exogenous retrovirus recently isolated from meat-type chicken lines, is similar to the viruses of these subgroups in group antigen but differs from them in envelope properties and has been assigned to a new subgroup, J. HPRS-103 has a wide host range in birds, and unlike other nontransforming ALSVs which cause late-onset B-cell lymphomas, HPRS-103 causes late-onset myelocytomas. Analysis of the sequence of an infectious clone of the complete proviral genome indicates that HPRS-103 is a multiple recombinant of at least five ALSV sequences and one EAV (endogenous avian retroviral) sequence. The HPRS-103 env is most closely related to the env gene of the defective EAV-E51 but divergent from those of other ALSV subgroups. Probing of restriction digests of line 0 chicken genomic DNA has identified a novel group of endogenous sequences (EAV-HP) homologous to that of the HPRS-103 env gene but different from sequences homologous to EAV and E51. Unlike other replication-competent nontransforming ALSVs, HPRS-103 has an E element in its 3' noncoding region, as found in many transforming ALSVs. A deletion found in the HPRS-103 U3 EFII enhancer factor-binding site is also found in all replication-defective transforming ALSVs (including MC29, which causes rapid-onset myelocytomas).

Alpharetrovirus↗

PubMed1995

Induction of AIDS by simian immunodeficiency virus from an African green monkey: species-specific variation in pathogenicity correlates with the extent of in vivo replication.

Previous studies suggested that simian immunodeficiency viruses isolated from African green monkeys (SIVagm) are relatively nonpathogenic. The report describes the isolation and biologic and molecular characterization of a pathogenic SIVagm strain derived from a naturally infected African green monkey. This virus induced an AIDS-like syndrome characterized by early viremia, frequent thrombocytopenia, severe lymphoid depletion, opportunistic infections, meningoencephalitis, and death of five of eight macaques within 1 year after infection. An infectious clone derived from this isolate reproduced the immunodeficiency disease in pig-tailed (PT) macaques, providing definitive proof of the etiology of this syndrome. Although the virus was highly pathogenic in PT macaques, no disease was observed in experimentally infected rhesus macaques and African green monkeys despite reproducible infection of the last two species. Whereas infection of PT macaques was associated with a high viral load in plasma, peripheral blood mononuclear cells, and tissues, low-level viremia and infrequent expression in lymph nodes of rhesus macaques and African green monkeys suggest that differences in pathogenicity are associated with the extent of in vivo replication. The availability of a pathogenic molecular clone will provide a useful model for the study of viral and host factors that influence pathogenicity.

Animals↗

PubMed1995

Spontaneous reversion of human immunodeficiency virus type 1 neutralization-resistant variant HXB2thr582: in vitro selection against cytopathicity highlights gp120-gp41 interactive regions.

Spontaneous revertants of the immune-selected variant HXB2thr582, which resists neutralization by certain conformationally dependent antibodies specific for the CD4-binding site on gp120 (such as F105), appeared after long-term culture in the absence of immune-selecting serum. Molecular analysis showed some of the viruses in the revertant stock contained a simple back mutation, whereas others retained the Thr-582 codon but contained a substitution of serine for phenylalanine in gp41 at position 673. Neutralization sensitivity to the selecting serum and to F105 of infectious clones containing either the back mutation or the compensatory mutation, HXB2thr582ser673, was confirmed. HXB2thr582-infected cells have a greater propensity for syncytium formation and single cell killing than do either the parental HXB2 or the revertant HXB2thr582ser673. This suggests that the revertant arose by selection in vitro for a less cytopathic virus. Our results link three envelope regions shown to influence virus-cell fusion as well as neutralization by antibody: the CD4-binding region, the leucine zipper domain, and a region hidden to antipeptide antibodies upon envelope oligomerization. Taken together they illustrate the functional importance of the gp120-gp41 interaction and emphasize the impact of the interplay between envelope regions on overall conformation and function and on recognition by neutralizing antibodies.

Amino Acid Sequence↗

PubMed1996

Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy.

Multidrug-resistant human immunodeficiency virus type 1 (HIV-1) strains with reverse transcriptase (RT) mutations at codons A62-->V, V75-->I, F77-->L, F116-->Y, and Q151-->M have been reported in patients receiving combination therapy with zidovudine (AZT) and didanosine (ddI). Infectious clones with each mutation alone, all five mutations together, and various combinations of mutations were created by site-directed mutagenesis. Mutation Q151-->M conferred partial resistance to AZT, ddI, zalcitibine, and stavudine, whereas a combination of four mutations conferred increased resistance to AZT, ddI, zalcitibine, and stavudine. The positions of residues 75, 77, and 151 in the three-dimensional crystal structure of HIV-1 RT suggest that these residues may affect the ability of the enzyme to discriminate between deoxynucleoside triphosphates and nucleoside analog RT inhibitors. Replication experiments showed that clones with mutation F77-->L but without V75-->I (HIV-1(77), HIV-1(77,151), and HIV-1(77,116,151) had attenuated growth compared with that of the original HIV-1NL4-3 strain and strains containing mutations at both positions 75 and 77 (HIV-1(75,77,151) and HIV-1(75,77,116,15)). Sequence analysis of viral RNA and proviral DNA from several patients indicated that RT mutations developed in a sequential and cumulative pattern over the course of a 2- to 4-year observation period. The present results suggest that drug resistance and viral replicative capacity both may play a role in selection of HIV-1 RT mutations.

Animals↗

PubMed1996

Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle.

Four calves were experimentally infected via aerosol with foot-and-mouth disease virus. Two were infected with a wild-type virus derived from a full-length infectious clone (A12-IC), and two were infected with a clone-derived virus lacking the leader gene (A12-LLV2), with euthanasia and tissue collection at 24 and 72 h postexposure (hpe). Clinical disease was apparent only in the animal given A12-IC and euthanized at 72 hpe. In situ hybridization revealed that the animal infected with A12-IC and euthanized at 24 hpe had abundant viral nucleic acid in the lung, present in clusters of positive cells in the respiratory bronchiolar epithelium and associated subepithelial regions. At 72 hpe in the A12-IC-infected calf, viral nucleic acid in the lung was present in interstitial areas, and in addition, viral nucleic acid was detectable in epithelial tissues around histologically apparent vesicles. In animals infected with A12-LLV2, viral nucleic acid was detectable in the lung at both 24 and 72 hpe, but staining revealed a more localized distribution with less nucleic acid than was found in animals given A12-IC. Therefore, it appears that after aerosol exposure to A12-IC, early replication is in the region of the lung, with subsequent dissemination to distal sites. In comparison, the A12-LLV2 virus is much less widely disseminated in the lung at 24 hpe, with no lesions or virus detectable in secondary sites at 72 hpe. The greatly reduced pathogenicity of A12-LLV2 may make it an excellent candidate for a modified live viral vaccine.

Animals↗

PubMed1997

Mutations in coat protein binding sites of alfalfa mosaic virus RNA 3 affect subgenomic RNA 4 accumulation and encapsidation of viral RNAs.

The 3'-untranslated regions (3'-UTRs) of the three RNAs of alfalfa mosaic virus (AMV) contain a specific binding site for coat protein (CP) and act as a promoter for minus-strand RNA synthesis by the purified AMV RNA-dependent RNA polymerase (RdRp) in an in vitro assay. Binding of CP to the viral RNAs is required to initiate infection. The sequence of the 3'-terminal 39 nucleotides of AMV RNA 3 can be folded into two stem-loop structures flanked by three single-stranded AUGC sequences and represents a CP binding site. Mutations in this sequence that are known to interfere with CP binding in vitro were introduced into an infectious clone of RNA 3, and mutant RNA transcripts were used as templates in the in vitro RdRp assay and to infect protoplasts and plants. Mutation of AUGC motif 2 or disruption of the stem of the 3'-proximal hairpin 1 interfered with CP binding in vitro but not with minus-strand promoter activity in vitro or replication of RNA 3 in vivo. However, hairpin 1 appeared to be essential for encapsidation of RNA 3. Reversion of three G-C base pairs in hairpin 1 had no effect on CP binding but interfered with minus-strand promoter activity in vitro and with RNA 3 replication in vivo. It is concluded that the viral RdRp and CP recognize different elements in the 3'-UTRs of AMV RNAs. Moreover, several mutations that interfered with CP binding in vitro interfered with the accumulation in vivo of RNA 4, the subgenomic messenger for CP, but not with the accumulation of RNA 3.

Alfalfa mosaic virus↗

PubMed1997

Infectivity enhancement by human immunodeficiency virus type 1 Nef is independent of its association with a cellular serine/threonine kinase.

Nef proteins from human immunodeficiency virus type 1 isolate SF2 (HIV-1SF2) and simian immunodeficiency virus isolate mac239 (SIVmac239) have been found to associate with a cellular serine/threonine kinase designated NAK. We have recently shown that the association of Nef with NAK is isolate dependent. To identify the structural basis for Nef-kinase association, several chimeric molecules were constructed between SF2 Nef (binding NAK) and 233 Nef (a primary isolate not binding NAK) and stably expressed in HuT-78 human T cells via retrovirus-mediated gene transfer. The Nef 233/SF2/SF2 chimera in which the N-terminal 37 amino acids of SF2 Nef were replaced by those of 233 Nef showed the same ability as SF2 Nef to bind NAK. The Nef 233/SF2/233 chimera in which the N-terminal 37 amino acids and the C-terminal 72 amino acids of SF2 Nef were replaced by corresponding sequences from 233 Nef completely lost the ability to associate with the kinase activity. Furthermore, replacement of the C-terminal 72 amino acids of 233 Nef with the equivalent SF2 sequence (chimera 233/233/SF2) fully restored kinase association to 233 Nef. These results suggest that (i) the core of Nef is not sufficient for NAK binding, (ii) the C terminus of SF2 Nef contains structural determinants important for association with NAK, and (iii) the failure of 233 Nef to bind NAK is due to a defect in its C terminus. Taking advantage of the C terminus of 233 Nef being nonfunctional and using an infectious clone of HIV-1SF2, we show that association with NAK is not required for Nef-mediated infectivity enhancement. While the strong and reproducible association of some Nef isolates with NAK has been clearly established, the role of NAK in Nef function remains to be fully elucidated.

Cell Line, Transformed↗