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J C Glorioso

Publications and source records attributed to J C Glorioso.

At least 91 records · Page 5Linked to original sources
PubMed1996

Gene transfer to neurons using herpes simplex virus-based vectors.

One important outgrowth of molecular medicine is the development of technologies for the transfer of therapeutic genes to cells in culture and tissues in vivo, which promises to revolutionize both experimental biomedical science and the clinical practice of medicine. Fundamental obstacles must still be overcome to create safe and efficient gene delivery vectors specifically designed for individual tissue types, and special strategies will be required for direct in vivo gene transfer to neurons because these cells are postmitotic and cannot be removed for transduction. Herpes simplex virus type 1 (HSV-1), a neurotropic virus that naturally establishes a latent state in neurons, has many unique features that make it suitable as a gene transfer vector for the nervous system. In this review we describe the molecular biology of HSV-1, strategies for reducing potential pathogenesis of the recombinant vector, and methods for expressing transgenes from the vector genome. Gene transfer experiments using recombinant HSV-1-based vectors and defective HSV-1 vectors (amplicons) for gene transfer are also described and evaluated in terms of efficiency and safety.

Alzheimer Disease↗
PubMed1996

In vivo transgene activation from an HSV-based gene therapy vector by GAL4:vp16.

Herpes simplex virus type 1 (HSV-1) has many attributes which make it attractive as a base for the development of vectors for the delivery of transgenes to the nervous system. In this report we describe the adaptation of the bipartite GAL4:VP16 transactivation system to replication-deficient HSV vectors. We demonstrate that the recombinant transactivator GAL4:VP16 produced from a replication-deficient HSV vector is capable of activating transcription of a reporter gene using a synthetic promoter consisting of GAL4 binding sites and the TATA box of the adenovirus E1b gene. Activation by vector produced GAL:VP16 was demonstrated with the recombinant promoter/reporter gene cassette in the infected cell chromosome, in the genome of a second virus infecting the same cells and with a single vector engineered to produce both GAL4:VP16 transactivator and to contain a recombinant promoter/reporter gene cassette. Furthermore, the double recombinant virus also produced the reporter gene product in neurons after direct intracranial inoculation into rat hippocampus. This system may be used to extend and improve promoter function in HSV gene transfer vectors in vivo.

Animals↗
PubMed1995

HSV as a gene transfer vector for the nervous system.

Gene therapy for diseases of the nervous system requires vectors capable of delivering the therapeutic gene into postmitotic cells in vivo. Herpes simplex virus type 1 is a neurotropic virus that naturally establishes latency in neurons of the peripheral nervous system. Replication defective HSV vectors have been developed; these are deleted for at least one essential immediate early regulatory gene, rendering the virus less cytotoxic, incapable of reactivation, but still capable of establishing latency. Foreign genes can be vigorously expressed from an HSV-based vector in a transient manner in brain and other tissues. Long-term but weak foreign gene expression may be achieved in the nervous system by exploiting the transcriptional control mechanisms of the natural viral latency active promoter. To meet the needs of specific applications, either highly active long-term or regulatable transgene expression will be needed, requiring further studies in order to design the appropriate latency-based promoter systems.

Gene Transfer Techniques↗
PubMed1995

Two herpes simplex virus type 1 latency-active promoters differ in their contributions to latency-associated transcript expression during lytic and latent infections.

Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.

Animals↗
PubMed1995

Development and application of herpes simplex virus vectors for human gene therapy.

Advances in understanding the molecular basis of human disease and the development of recombinant DNA methods is rapidly creating new means of disease diagnosis and treatment. Among the most revolutionary developments are technologies for transfer of therapeutic genes to the human body to treat both inherited and acquired disease. Gene therapy offers considerable promise for ameliorating otherwise intractable diseases such as immunopathological conditions, cancer, heart disease, and various metabolic and neurodegenerative syndromes. To fulfill this promise, more efficient and effective methods of gene delivery and appropriate gene expression must be developed. The lack of such techniques is currently the most significant impediment to the use of genetic therapy. Both viral and nonviral delivery systems are under development for specific gene-therapy applications. Herpes simplex virus (HSV) represents a novel vector system for gene delivery to the nervous system and other tissues. HSV is able to establish latency in nondividing neuronal cells in which genomes persist long-term but do not integrate or alter host-cell metabolism and that carry a promoter system uniquely capable of escaping repression that shuts off the expression of HSV-lytic genes during latency. This review examines efforts to create defective HSV vectors that are safe, noncytotoxic, and applicable to the treatment of cancer and diseases affecting peripheral nerves. Perhaps the most important use of HSV vectors will be for the treatment of neurodegenerative diseases of the brain, but additional studies are required to improve the design of promoters to ensure regulatable or effective levels of therapeutic gene expression.

Brain Diseases↗
PubMed1995

Herpes simplex virus type 1 vector mediated gene transfer to muscle.

Herpes simplex virus type 1 (HSV-1) has a broad host range although in natural human infections the virus is neurotropic, establishing latent infections in sensory neurons where the viral DNA persists as an intact episome. The establishment of latency does not depend on viral replication functions, suggesting that infection of non-neuronal cells, including tissue of myogenic origin, by replication defective mutants may result in genome persistence in a similar episomal state. In this report a replication defective HSV-1 recombinant vector containing the beta-galactosidase reporter gene under transcriptional control of the strong human cytomegalovirus immediate-early gene promoter (HCMV IEp-lacZ) was used to infect muscle cells in vitro and in vivo. This replication defective mutant virus (d120), deleted for both copies of the essential immediate-early gene (ICP4) and thus incapable of expressing early and late viral genes, displayed highly reduced cytotoxicity in myogenic cells. This vector infected both myoblasts and myotubes in culture with transgene expression persisting for at least 8 days. The transduction efficiency in myotubes was similar to myoblasts at several multiplicities of infection (MOIs), suggesting that HSV could infect differentiated muscle fibers and that myoblast differentiation would neither prevent expression of the cellular receptor(s) for the virus nor inhibit viral penetration. Direct inoculation of mouse muscle fibers in vivo with 10(6) to 10(8) plaque forming units (p.f.u.) of vector was sufficient to transduce significant numbers of muscle fibers in newborn mice and some fibers in adult normal and mdx mice. These results suggest that recombinant HSV-1 vectors may be useful for gene transfer to muscle.

Animals↗
PubMed1995

Antiviral activity of herpes simplex virus vectors expressing murine alpha 1-interferon.

Mutant herpes simplex virus type I (HSV-1) vectors were engineered to express murine alpha 1 interferon (IFN) and assessed for their ability to inhibit the replication of challenge viruses in infection of monolayer cell cultures. The alpha 1 IFN gene was placed under control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) region in the thymidine kinase (tk) locus of both the wild-type HSV-1 strain KOS and the replication-defective KOS mutant dl20, in which both copies of the ICP4 immediate-early (IE) gene are deleted. To evaluate IFN expression, vector-infected cell culture media from epithelial, fibroblast and neuronal cells were assayed for alpha IFN release. These cells did not secrete detectable levels of alpha IFN when infected with control KOS or d120-based viruses. Cells infected at a multiplicity of infection (MOI) of 10 with either RSV-LTR-IFN vector produced from approximately 12 to 100 U of alpha 1-IFN per millilitre of culture fluid in 24 h, as determined by a standard vesicular stomatitis virus (VSV) replication inhibition assay. Vector-derived alpha 1-IFN expression did not inhibit the growth of the KOS-RSV-IFN virus in IFN sensitive cell lines. However, pretreatment of murine L cells with IFN produced from the RSV-IFN vectors blocked the replication of HSV-1. Additionally, L cells infected with d120-RSV-IFN (at an MOI of 0.5 to 0.06) were protected from superinfection with VSV. Thus, pre-infection of a small percentage of cells with a nonreplicating HSV-IFN expression vector provided complete protection of tissue culture cell monolayers from the cytopathic effect of a challenge virus infection. These vectors should be useful for in vivo analysis of the antiviral potential of IFN expression from within the nervous system.

Animals↗
PubMed1994

Gene transfer to brain using herpes simplex virus vectors.

Herpes simplex virus type 1 represents an ideal candidate for development as a vehicle for gene transfer to postmitotic neurons of the central nervous system. The natural biology of this virus makes it well suited for this purpose as it is capable of infecting a variety of neuronal cell types in the brain where the viral genome can persist indefinitely in a latent state. In latency, the viral lytic genes are transcriptionally silent and a unique set of latency-associated transcripts are expressed. Two impediments to using herpes simplex virus vectors must be overcome: (1) A noncytotoxic mutant virus backbone must be engineered, and (2) a suitable promoter-regulator that stably expresses foreign genes from the vector genome during latency must be constructed. Deletion of specific immediate early genes from the vector can render the virus nontoxic to neurons in culture and in vivo following stereotactic inoculation into specific regions of the brain. Because these viruses cannot replicate, they enter latency on infection of central nervous system neurons. A number of viral and cellular promoters have been tested for their ability to express genes during latency. Strong viral promoters and neurospecific promoters display transient activity. Although the promoter regions for the latency-associated transcripts are highly active in the peripheral nervous system, they show low-level but persistent activity in the brain. Experiments are in progress to exploit RNA polymerase III gene promoters or novel recombinant promoters capable of auto-inducing their own expression in order to increase gene expression during latency in brain neurons.

Animals↗
PubMed1994

A genetic selection method for the transfer of HSV-1 glycoprotein B mutations from plasmid to the viral genome: preliminary characterization of transdominance and entry kinetics of mutant viruses.

A genetic selection procedure has been devised to transfer mutations from a plasmid to the viral genome. Reagents were constructed so that the recombination events that occur during cotransfection rescue a mutation in a recipient viral genome and, simultaneously, transfer a mutation in an adjacent (target) gene to the viral genome. In the example presented here the two adjacent genes are essential for viral replication, so that a transformed cell line that expresses both genes and another cell line that expresses only the target gene are required. Ideally the recipient viral genome should be deleted for the entire target gene, and the deletion should extend a short distance into the adjacent gene. In the present study UL27 (glycoprotein gB) of herpes simplex virus type 1 (KOS) is the target gene for mutation transfer and the upstream gene UL28, which specifies the ICP18.5 polypeptide, is the marker-rescue gene. A recipient virus that was deleted for DNA sequences encoding the C-terminal 74 residues of ICP18.5 and the N-terminal 711 residues of gB was constructed. All of the gB mutant plasmids used overlap the ICP18.5 deletion, and therefore, recombination events that rescue the ICP18.5 deletion must transfer a gB mutation present in codons 1-711 of the rescuing plasmid. Recombinant viruses that contain the gB mutation, unlike the parental gB-/18.5- virus, will grow on cells that express only gB (D6). This procedure has been used to transfer insertion, deletion, and chain termination mutations into the gB gene of the KOS genome. Southern blot analysis confirmed the transfer of the mutations to the viral genome. Analysis of radioactively labeled, infected cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), before and after immune precipitation with gB-specific antibodies, confirmed the presence of the mutant polypeptides in the lysates. The incorporation of the mutant polypeptides into the envelopes of purified released viruses was measured. The yield of plaque forming units per cell was determined from single step growth curves after infection of permissive D6 cells. Virus yield, a measure of transdominance, varied for the different mutants. The maximum reduction in virus yield observed was sixfold relative to the wild-type virus. Transdominance was a result of both the lack of glycoprotein processing and the presence of gB-oligomerization sites. A reduced yield was also observed for a previously isolated gB-null mutant virus, K082. The amount of gB produced in D6 cells, infected with K082, was less than that produced in KOS infected cells, which may account for the reduced yield.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Line↗
PubMed1994

Localization of a functional site on herpes simplex virus type 1 glycoprotein C involved in binding to cell surface heparan sulphate.

The amino acid residues critical for interaction between herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) and cell surface heparan sulphate (HS) were localized to two separate regions within antigenic site II of this glycoprotein. These amino acids were Arg-143, Arg-145, Arg-147 and Thr-150 in one region and Gly-247 in the other. This conclusion is based on the following observations. (i) Monoclonal antibodies defining gC-1 antigenic site II, and not those reactive with antigenic site I, inhibited HSV-1-induced haemagglutination and virus binding to susceptible cells. (ii) A number of HSV-1 mar mutants, altered at these critical residues, were impaired in attachment to cells. (iii) Synthetic peptides, corresponding to these two regions inhibited virus attachment to cells and infectivity. In addition these peptides were found to agglutinate red blood cells. This agglutination was inhibited by soluble HS, and was prevented by the pretreatment of red blood cells with heparitinase suggesting that cell surface HS was a site of peptide binding. The same was observed with the polycationic substances neomycin and poly-L-lysine. In conclusion, we propose that the regions of gC-1 represented by the HS-binding peptides may form a functional site of a polycationic nature, active in attachment to the polyanionic glycosaminoglycan chain of cell surface HS.

Amino Acid Sequence↗
PubMed1994

Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain.

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.

Animals↗
PubMed1994

A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats.

Herpes simplex virus type 1 (HSV-1) expresses a unique series of RNA molecules, the latency-associated transcripts or LATs, during latent infection of neuronal tissues. Previous studies by others have described a TATA box-containing latency-active promoter, referred to here as LAP1, located approximately 700 bp upstream of the 5' end of the major 2.0-kb LAT. In this report, transient gene expression assays were employed to identify a second, novel latency-active promoter (LAP2) present within a region downstream of LAP1 and 5' proximal to the major 2.0-kb LAT. In contrast to LAP1, this promoter lacks a TATA box but possesses cis-acting regulatory elements and other features frequently observed within eukaryotic housekeeping gene promoters. Unlike most other HSV promoters, LAP2 was down-regulated by the viral transcriptional activators ICP4 and ICP0. The majority of LAP2-positive regulatory elements were located within sequences from -257 to -58 relative to the 5' end of the 2.0-kb LAT, and the basal promoter mapped within sequences from -14 to +28. RNase protection experiments demonstrated that chimeric LAT-chloramphenicol acetyltransferase transcripts produced in the transient assays initiated at or near the 5' end of the major 2-kb LAT. Tn5 insertional mutagenesis of the ICP4 regulatory gene determined that down-regulation of LAP2 required the ICP4 transactivating domain and targeted the minimal promoter region as the site of action by ICP4. Replicating recombinant viruses containing a LAP2-lacZ reporter gene cassette in an ectopic site (glycoprotein C locus) were shown to be active in mouse trigeminal ganglia. Taken together, these experiments suggest that the LAT region of the HSV-1 genome contains at least two latency-active promoters which may play different roles in expressing the various LATs. Alternatively, these promoters may comprise a larger promoter-regulatory complex which may influence transcription during latency.

Animals↗
PubMed1994

Herpes simplex virus vectors and gene transfer to brain.

Experiments are in progress to engineer herpes simplex virus type 1 as a gene transfer vector for the nervous system. This virus is well suited for this purpose due to its ability to persist for long periods in a latent state in neurons wherein lytic genes are inactive and a unique viral promoter is capable of inducing transcription from the latent viral genome. The virus can be highly cytotoxic for neurons, however, and thus genes which initiate the lytic cycle must be deleted from the genome in order to force the virus into latency. Candidate genes include the immediately early functions ICP0, ICP27, ICP4 and the virus host shut-off function. The design of a suitable promoter regulator capable of expressing foreign genes during latency must also be developed. Many promoters including strong viral promoters and neuronal-specific cellular promoters have proved to be inadequate due to the transient nature of their activity from the viral vector genome and the viral latency promoter region appears to be weakly active in brain. Current efforts concern the development of auto-regulatable promoters which can remain active even if the viral genome is bound by chromatin.

Base Sequence↗
PubMed1993

Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.

Animals↗
PubMed1993

The RR1 gene of herpes simplex virus type 1 is uniquely trans activated by ICP0 during infection.

As has been demonstrated for herpes simplex virus type 2, we show in this report that the herpes simplex virus type 1 ribonucleotide reductase large subunit (RR1) gene is trans activated in transient transfection assays by VP16 and ICP0 but not by ICP4. Deletion analysis demonstrated that responsiveness to induction to VP16 resides in an octamer/TAATGARAT sequence of the RR1 promoter and that the TATA box alone is sufficient to provide induction by ICP0. The induction of the RR1 gene by ICP0 but not by ICP4 suggested that it might be possible to identify the cis-acting element(s) responsive to ICP4 in an ICP4-inducible promoter. To this end, a series of chimeric promoters containing various portions of the regulatory sequences of the RR1 promoter and thymidine kinase (TK) promoter were constructed. The TK promoter is trans activated by both ICP0 and ICP4 in transient transfection assays and by ICP4 in infection. The data show that replacing the RR1 TATA region with the TK TATA region permits ICP4 inducibility even if the rest of the RR1 promoter elements remain intact. To test whether the RR1 gene is induced by ICP0 during infection, four mutant viruses were constructed. (i) TAATGARAT+ has the wild-type RR1 promoter driving chloramphenicol acetyltransferase (CAT) and the RR2 promoter driving the lacZ gene. The RR2 gene codes for the small subunit of the ribonucleotide reductase and is expressed as a beta gene. (ii) TAATGARAT- has a triple-base change in the octamer/TAATGARAT element which renders it unresponsive to VP16 trans activation, eliminating that portion of the activation of the RR1 gene. (iii) TAATGARAT- delta alpha 0 has a deletion of the alpha 0 gene. (iv) TAATGARAT- delta alpha 4 has a deletion of the alpha 4 gene. Infections were carried out in Vero cells at a multiplicity of infection of 10 per cell; cells were assayed for CAT and beta-galactosidase (beta-Gal) activities and for virus yields. The first two infections gave strong CAT and beta-Gal activities and high yields of progeny virus. Infection with the third virus showed no CAT activity but did produce high levels of beta-Gal activity and virus progeny. The fourth infection resulted in strong CAT activity but no beta-Gal activity or progeny virus. The data demonstrated that the RR1 promoter was activated in the absence of ICP4 but not in the absence of ICP0 in these infections.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Mutations in herpes simplex virus type 1 genes encoding VP5 and VP23 abrogate capsid formation and cleavage of replicated DNA.

The herpes simplex virus type 1 capsid is composed of seven capsid proteins which are termed VP5, VP19c, VP21, VP22a, VP23, VP24, and VP26. Major capsid protein VP5 is encoded by the gene UL19. UL18, whose transcript is 3' coterminal with that of VP5, specifies capsid protein VP23. Vero cell lines have been isolated that are transformed with either the BglII N (UL19) or EcoRI G (UL16 to UL21) fragment of KOS. These cell lines, selected for the ability to support the replication of a temperature-sensitive VP5 mutant, were used to isolate VP5 and VP23 null mutants. The mutations in VP5 (K5 delta Z) and VP23 (K23Z) were generated by insertion of the lacZ gene at the beginning of the coding sequences of the genes. Both mutants failed to form plaques on the nonpermissive cell line, and therefore, VP23, like VP5, is an essential gene product for virus replication. Both mutants expressed wild-type levels of infected-cell proteins upon infection of permissive and nonpermissive cell lines. However, the VP5 (150-kDa) and VP23 (33-kDa) polypeptides were absent in lysates prepared from K5 delta Z- and K23Z-infected Vero cells, respectively. No capsid structures were observed by electron microscopic analysis of thin sections of K5 delta Z- and K23Z-infected Vero cells. Following sedimentation of lysates from cells infected by the mutants, capsid proteins were not observed in the fractions where capsids normally sediment. The amounts of DNA replicated in the VP5 and VP23 mutant and in KOS-infected Vero cells were the same as in permissive cells. However, genomic ends were not evident in Vero cells infected with the mutants, suggesting that the DNA remains in concatemers and is not processed into unit length genomes.

Animals↗