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PubMed1998

Delayed progression of murine AIDS in C57BL/6 mice pre-immunized with a highly antigenic 10-mer peptide encoded by the murine AIDS defective virus gag p12 gene.

C57BL/6 (B6) mice were immunized with a highly antigenic 10-mer peptide (P12-10), which is encoded by the murine AIDS (MAIDS) defective virus gag p12 gene, emulsified in incomplete Freund's adjuvant (ICFA). One week later, the mice were inoculated with the MAIDS virus to see if the immunization affects progression of MAIDS. It was demonstrated that the immunization significantly delayed progression of MAIDS, although it failed to induce appreciable cytotoxic T lymphocyte (CTL) responses against the P12-10 antigen. In contrast, immunization of B6 mice with the P12-10 coupled with liposome induced substantial CTL responses but failed to protect the mice against MAIDS development. This segregation between CTL activity and in vivo protection efficacy might be worth considering when we exploit vaccines for augmenting cellular immunity mediated by CD8+ T cells.

Animals↗
PubMed1996

Evidence that the murine AIDS defective virus does not encode a superantigen.

The T-cell receptor repertoire was analyzed in C57BL/6 mice upon infection with helper-free stocks of the pathogenic murine AIDS (MAIDS) defective virus in order to demonstrate if, as previously reported, this virus encodes a superantigen. A polyclonal T-cell stimulation involving T cells expressing multiple V beta subsets occurred within the first week of infection, while late in the disease we could note only a 50% deletion of V beta 5 CD8+ cells. Transfection of the MAIDS virus genomic DNA into fibroblasts and B cells expressing major histocompatibility complex class II molecules failed to show any stimulation of cells expressing the specific V beta (V beta 5) previously reported to respond to MAIDS virus-infected cells. In addition, mice lacking V beta 5 cells did not show any significant decrease in susceptibility to the disease compared with mice expressing V beta 5 and bred on the same genetic background. Our in vivo and in vitro results fail to demonstrate a role for a superantigen encoded by the MAIDS defective viral genome in the pathogenesis of MAIDS.

Animals↗
PubMed2006

Superinfection of human immunodeficiency virus type 1 (HIV-1) to cell clone persistently infected with defective virus induces production of highly cytopathogenic HIV-1.

Superinfection with human immunodeficiency virus type 1 (HIV-1) in human subjects, defined as reinfection with a heterologous strain of HIV-1, has become a topic of great interest. To illustrate the significance of this occurrence, we performed HIV-1 superinfection of L-2 cells, which were isolated from MT-4 cells persistently infected with subtype B HIV-1 as a cell clone continuously producing defective HIV-1 particles. L-2 cells carrying provirus with a one-base insertion in the pol protease were superinfected with HIV-1 derived from primary isolates of subtype B or CRF01_AE. The kinetics of the superinfection in L-2 were very slow compared with those of primary infections in MT-4. Interestingly, L-2 shifted after superinfection to become a producer of highly cytopathogenic HIV-1. Molecular characterization revealed that superinfection occurred in only about 10% of the CRF01_AE-superinfected L-2, which carried provirus of both subtypes and produced viral particles containing genomic RNA of both subtypes. Surprisingly, such cytopathogenic HIV-1 showed predominantly the original subtype B phenotype. Thus, the mechanism of the production of cytopathic HIV-1 seemed to be mediated by trans complementation with pol products of superinfected CRF01_AE. These findings suggest the significance of long-lived infected cells as recipients for superinfection that could result in the generation of new HIV-1 variants with high virulence in patients who are off therapy or do not adhere to treatment, and may indicate the need for precautions against such superinfection.

Cell Line↗
PubMed1991

Expression of defective virus and cytokine genes in murine AIDS.

A syndrome characterized by severe immunodeficiency and lymphoproliferation develops in susceptible strains of mice infected with a mixture of murine leukemia viruses (MuLVs) designated LP-BM5 MuLV. The etiologic agent in this mixture has been shown to be a replication-defective virus (BM5d) with a 4.8-kb genome that required replication-competent helper viruses, primarily ecotropic (BM5e), for cell-to-cell spread in the host. In the present study, we studied the expression of BM5d and BM5e in tissues of infected mice at various times after inoculation in relation to the expression of cytokine genes that may contribute to the pathogenesis of this disorder. Northern (RNA) analysis of total RNA showed that BM5d was expressed at significant levels in lymphoid tissues within 1 week of infection and that the levels of expression increased with time after inoculation. By 16 weeks postinfection, BM5d was expressed in all tissues examined. Expression of BM5e was relatively more restricted to lymphoid tissues and was detected at lower levels than expression of BM5d at early times after infection, but this virus was expressed in all tissues by 16 weeks. Infection with the virus mixture was associated with constitutive expression of tumor necrosis factor in all tissues examined and of interleukin-1 (IL-1) in lymphoid tissues within 1 week of infection, and at later times with widespread expression of these cytokines and gamma interferon. Also, the levels of interferon regulatory factor 1 mRNA were significantly increased in all infected tissues during the infection. In contrast, expression of IL-3, IL-4, IL-5, and IL-6 was not detectable by Northern analysis of the respective mRNAs in any infected tissue at early or late times postinfection.

Animals↗
PubMed2001

Green fluorescent protein expression from recombinant lettuce infectious yellows virus-defective RNAs originating from RNA 2.

Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1.

Blotting, Northern↗
PubMed1965

ADENOVIRUS-ASSOCIATED DEFECTIVE VIRUS PARTICLES.

Small, DNA-containing particles were separated from preparations of a simian adenovirus. These particles differed antigenically from the adenovirus. Replication of the particles in cell cultures was obtained only when theywere inoculated simultaneously with adenoviruses. This suggests that these adenovirus-associated particles behave as defective viruses.

Adenoviridae↗
PubMed1991

An enhancer variant of Moloney murine leukemia virus defective in leukemogenesis does not generate detectable mink cell focus-inducing virus in vivo.

Moloney murine leukemia virus (Mo-MuLV) induces T-cell lymphoma when inoculated into neonatal mice. This is a multistep process. Early events observed in infected mice include generalized hematopoietic hyperplasia in the spleen and appearance of mink cell focus-inducing (MCF) recombinants; end-stage tumors are characterized by insertional proviral activation of protooncogenes. We previously showed that an Mo-MuLV enhancer variant, Mo+PyF101 Mo-MuLV, has greatly reduced leukemogenicity and is deficient in induction of preleukemic hyperplasia. In this report, we have examined Mo+PyF101 Mo-MuLV-inoculated mice for the presence of MCF recombinants. In contrast to wild-type Mo-MuLV-inoculated mice, Mo+PyF101 Mo-MuLV-inoculated mice did not generate detectable MCF recombinants. This failure was at least partly due to an inability of the MCF virus to propagate in vivo, since a molecularly cloned infectious Mo+PyF101 MCF virus did not replicate, even when inoculated as a Mo+PyF101 Mo-MuLV pseudotype. These results show that the leukemogenic defect of Mo+PyF101 Mo-MuLV is associated with its inability to generate MCF recombinants capable of replication in vivo. This, in turn, is consistent with the view that MCF recombinants play a significant role in Mo-MuLV-induced disease and, in particular, may play a role early in the disease process.

Animals↗
PubMed2004

Host-specific generation and maintenance of Tomato bushy stunt virus defective interfering RNAs.

The accumulation of Tomato bushy stunt virus (TBSV) defective interfering RNAs (DIs) has been observed in several species of plants, but the involvement of host-specific processes and the functional role of DIs are still poorly understood. In this study, the accumulation of DIs was compared after several passages of TBSV through Nicotiana benthamiana and pepper (Capsicum annuum). As anticipated, passages of wild-type TBSV through N. benthamiana resulted in the accumulation of significant levels of TBSV DIs, which caused symptom attenuation and prevented the plants from lethal necrosis. On the contrary, TBSV infection of pepper plants caused severe local and systemic chlorosis, but continuous virus passages did not result in detectable levels of DIs accumulation. In addition, the inoculation of pepper plants with a mixture of helper virus and DI either from in vitro generated transcripts or from infected N. benthamiana did not yield DI in upper pepper leaves. Our cumulative results suggest that complex host-specific determinants play an important role in TBSV DI generation and their subsequent maintenance and accumulation.

Capsicum↗
PubMed2001

Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain.

A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.

Animals↗
PubMed1988

Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine.

By starting from a thrice-purified wild-type measles virus plaque, the generation of detectable subgenomic RNAs was achieved within a series of five serial infections of Vero cells. The evolution of these subgenomic RNAs was followed for seven serial passages and ended with the preparation of a highly interfering viral stock. On the other hand, the detection of discrete subgenomic RNAs was achieved during the first infection of Vero cells with at least one of three measles virus vaccine preparations tested. These subgenomic RNAs, which interfered very efficiently with the replication of the endogenous standard genomes upon vaccine infection but showed a moderate interfering activity with a standard virus stock derived by plaque purification from the vaccine preparation, resulted from the presence of defective interfering particles in the vaccine preparation. The relevance of this finding for the attenuation, stability, and potential capacity for persistent infection of such a vaccine is discussed.

Chromosome Mapping↗
PubMed1983

Replication of vesicular stomatitis virus defective interfering particle RNA in vitro: transition from synthesis of defective interfering leader RNA to synthesis of full-length defective interfering RNA.

The replication of the RNA of vesicular stomatitis virus (VSV) defective interfering (DI) particles was established in a defined cell-free system. The transition from synthesis of only the DI-leader RNA to replication of the full-length DI RNA was effected in the system by newly synthesized VSV proteins and occurred in the absence of VSV helper virus. Both positive- and negative-polarity full-length DI RNA were synthesized. Furthermore, the products of RNA replication associated with newly synthesized viral proteins to form complexes that were indistinguishable from authentic DI particle nucleocapsids on the basis of buoyant density and resistance to ribonuclease digestion. The DI-leader RNA did not form ribonuclease-resistant structures. We conclude that this in vitro system successfully executes many of the reactions of VSV DI particle replication and assembly.

Capsid↗
PubMed1966

Replication in simian cells of defective viruses in an SV40-adenovirus "hybrid" population.

Butel, Janet S. (Baylor University College of Medicine, Houston, Tex.), and Fred Rapp. Replication in simian cells of defective viruses in an SV40-adenovirus "hybrid" population. J. Bacteriol. 91:278-284. 1966.-An SV40-adenovirus type 7 "hybrid" virus population, previously shown to contain two viruses capable of complementation in green monkey kidney (GMK) cells, has a growth cycle in GMK cells similar to that of adenovirus type 7 in the presence of SV40. Extending previous preliminary results, the addition of adenovirus types 2, 7, or 12 to monolayers of GMK cells enhanced plaque formation by the SV40-adenovirus hybrid by as much as 200-fold. The terminal enhanced plaques, initiated by the hybrid in the presence of helper adenovirus, were found to contain progeny which could induce the synthesis of SV40 tumor antigen but which were coated with the protein of the helper adenovirus, type 2, 7, or 12, respectively. The particle carrying the SV40 tumor antigen determinant, named PARA, is defective in that it cannot direct the synthesis of capsid protein; information for the coat for PARA is supplied by the adenovirus. One-step growth curves of the hybrid virus population in monkey cells revealed that synthesis of both types of particles, adenovirus and PARA, proceeds at a similar rate, with a latent period of 16 to 20 hr being followed by an exponential increase in titer during the following 20 hr. Maximal titers for both particles were obtained 48 hr after inoculation of the cultures. Neither the PARA nor the adenovirus component replicated in GMK cells in the absence of the other.

Adenoviridae↗
PubMed1994

Myristylation of Pr60gag of the murine AIDS-defective virus is required to induce disease and notably for the expansion of its target cells.

Murine AIDS (MAIDS) is characterized by severe lymphadenopathy and splenomegaly. The proliferation of the infected target B cells is also an important manifestation of the disease (M. Huang, C. Simard, D. G. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). The etiologic agent of MAIDS is a defective murine leukemia virus that is deleted of most of its pol and env genes and appears to encode a single protein, the Gag precursor Pr60gag protein. Pr60gag is myristylated and attached to the plasma membrane. To study the role myristylation on the function of Pr60gag, we have generated a myristylation-negative (Myr-) mutant of the MAIDS defective virus. We found that Myr- Pr60gag interacted less tightly with the plasma membrane. In addition, the Myr- MAIDS defective virus mutant was unable to induce expansion of infected cells and was nonpathogenic. These results emphasize the essential role of Pr60gag in the disease process. Our data also suggest that Pr60gag, once recruited to the cell membrane through its myristylation, interacts with other membrane-bound effectors to send signals to induce proliferation of the infected cells and to initiate immune dysfunctions.

Animals↗
PubMed1983

Sequence analysis of two mutants of Sindbis virus defective in the intracellular transport of their glycoproteins.

We have sequenced the complementary DNA corresponding to the genes encoding the viral glycoproteins of ts10 and ts23, mutants of Sindbis virus defective in the intracellular transport of their glycoproteins, and of revertants of these mutants. These studies have been augmented by direct amino acid sequencing of the amino-terminal regions of the glycoproteins of several virus strains. By comparing the deduced amino acid sequence with that of Sindbis HR virus, the parental strain of these mutants, and with the sequence of the revertants, we found ts23 to have a double mutation in glycoprotein E1, while ts10 was a single mutant in the same glycoprotein. In each case reversion to temperature insensitivity occurred by changes at the same site as the mutation, in two cases restoring the original amino acid and in the third case substituting an homologous amino acid (arginine in place of lysine). The three mutations were far apart from each other in the protein, suggesting that the three-dimensional conformation is very important for the correct migration of the glycoproteins from the rough endoplasmic reticulum to the plasma membrane. The sequence data also reveal that a number of other changes have occurred in the various virus strains during mutagenesis or passage.

Amino Acid Sequence↗
PubMed1999

Ebola virus defective interfering particles and persistent infection.

Ebola virus (Zaire subtype) is associated with high mortality disease outbreaks that commonly involve human to human transmission. Surviving patients can show evidence of prolonged virus persistence. The potential for Ebola virus to generate defective interfering (DI) particles and establish persistent infections in tissue culture was investigated. It was found that serial undiluted virus passages quickly resulted in production of an evolving population of virus minireplicons possessing both deletion and copyback type DI genome rearrangements. The tenth undiluted virus passage resulted in the establishment of virus persistently infected cell lines. Following one or two crises, these cells were stably maintained for several months with continuous shedding of infectious virus. An analysis of the estimated genome lengths of a selected set of the Ebola virus minireplicons and standard filoviruses revealed no obvious genome length rule, such as "the rule of six" found for the phylogenetically related Paramyxovirinae subfamily viruses. Minimal promoters for Ebola virus replication were found to be contained within 156 and 177 nucleotide regions of the genomic and antigenomic RNA 3' termini, respectively, based on the length of authentic termini retained in the naturally occurring minireplicons analyzed. In addition, using UV-irradiated preparations of virus released from persistently infected cells, it was demonstrated that Ebola virus DI particles could potentially be used as natural minireplicons to assay standard virus support functions.

Animals↗
PubMed2000

Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs.

Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156-3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3'- and 5'-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5'-proximal region was found to be approximately 100-fold lower than that in the 3'-proximal part of the genome.

Animals↗
PubMed1998

Characterization of Toscana virus-defective interfering particles generated in vivo.

Toscana (TOS) virus stocks strongly interfering with standard virus replication were obtained by sequential passages of virus in suckling mouse brain. Characterization of viral RNAs in these stocks showed the presence of a heterogeneous population of defective RNA molecules derived from the L genomic segment, in both nucleocapsid (NC) and messenger RNAs, suggesting that these molecules could be replicated, assembled, and transcribed. Subgenomic RNAs from the L segment but not from the S or M segments were found in cells infected with these stocks. Defective RNA molecules interfered with virus replication and retained 5' and 3' genomic termini. Nucleotide sequence analysis of some cloned defective interfering (DI) RNAs revealed they contained one or more internal deletions reducing their length to 7-13% of the full-length L segment. An identical sequence motif, of variable length, was found at both terminal sites of the RNA junction on standard L sequences. This motif was retained only in one copy in the subgenomic RNA. These results are consistent with the generation of TOS virus DI particles in vivo and suggest that the defective genomic RNAs could be generated by polymerase jumping from a sequence to an identical one spatially closed because of the RNA structure.

Animals↗
PubMed1985

The alpha sequence of the cytomegalovirus genome functions as a cleavage/packaging signal for herpes simplex virus defective genomes.

Although herpes simplex virus (HSV) 1 and human cytomegalovirus (CMV) differ remarkably in their biological characteristics and do not share nucleotide sequence homology, they have in common a genome structure that undergoes sequence isomerization of the long (L) and short (S) components. We have demonstrated that the similarity in their genome structures extends to the existence of an alpha sequence in the CMV genome as previously defined for the HSV genome. As such, the alpha sequence is predicted to participate as a cis-replication signal in four viral functions: (i) inversion, (ii) circularization, (iii) amplification, and (iv) cleavage and packaging of progeny viral DNA. We have constructed a chimeric HSV-CMV amplicon (herpesvirus cis replication functions carried on an Escherichia coli plasmid vector) substituting CMV DNA sequences for the HSV cleavage/packaging signal in a test of the ability of this CMV L-S junction sequence to provide the cis signal for cleavage/packaging in HSV 1-infected cells. We demonstrate that the alpha sequence of CMV DNA functions as a cleavage/packaging signal for HSV defective genomes. We show the structure of this sequence and provide a functional demonstration of cross complementation in replication signals which have been preserved over evolutionary time in these two widely divergent human herpesviruses.

Animals↗