Characterization of an infectious cDNA copy of the genome of a naturally occurring, avirulent coxsackievirus B3 clinical isolate

Group B coxsackieviruses (CVB) cause numerous diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes. To date, infectious cDNA copies of CVB type 3 (CVB3) genomes have all been derived from pathogenic virus strains. An infectious cDNA copy of the well-characterized, non-pathogenic CVB3 strain GA genome was cloned in order to facilitate mapping of the CVB genes that influence expression of a virulence phenotype. Comparison of the sequence of the parental CVB3/GA population, derived by direct RT-PCR-mediated sequence analysis, to that of the infectious CVB3/GA progeny genome demonstrated that an authentic copy was cloned; numerous differences were observed in coding and non-coding sequences relative to other CVB3 strains. Progeny CVB3/GA replicated similarly to the parental strain in three different cell cultures and was avirulent when inoculated into mice, causing neither pancreatitis nor myocarditis. Inoculation of mice with CVB3/GA protected mice completely against myocarditis and pancreatitis induced by cardiovirulent CVB3 challenge. The secondary structure predicted for the CVB3/GA domain II, a region within the 5′ non-translated region that is implicated as a key site affecting the expression of a cardiovirulent phenotype, differs from those predicted for cardiovirulent and pancreovirulent CVB3 strains. This is the first report characterizing a cloned CVB3 genome from an avirulent strain.

Two Cellular Mechanisms for Gamma-Globin Gene and Protein Silencing in Humans.

Although the genetic processes responsible for gamma-globin gene and protein silencing are not known, the prevailing model is that gamma-globin silencing results from a gradual change within a single hematopoietic cell lineage that is governed by intrinsic properties of the cells. In order to provide a more complete characterization of the silencing phenomenon, we studied globin expression patterns directly from clinical samples using single-cell, quantitative PCR, and globin protein phenotyping. We collected blood samples from untransfused donors: umbilical cords (n=3), infants (n=11; ages 1 day to 35 months), and adults (n=3). All samples were maintained at 4°C and analyzed within 72 hours. Flow cytometry (30,000 cells per donor) and HPLC analyses were used for globin protein phenotyping. For globin gene expression, we identified reticulocytes using a strategy that required no membrane permeabilization, and sorted them as single cells directly into lysis buffer. Oligo-dT reverse transcription of mRNA was followed by real-time PCR quantitation. Globin cDNA copy numbers were calculated using standard curves from serial dilutions of a plasmid DNA. We analyzed approximately 1000 single-cell quantitative PCR amplifications for gamma- and beta-globin gene expression. In cord blood, we detected both gamma- and beta-globin gene expression in 97.4% (112/115) of the reticulocytes. The average gamma-globin cDNA copy number was 1870±1390 copies, compared with an average beta-globin cDNA copy number of 2181±2138 copies per reticulocyte. HbF and HbA were also detected in >95% of the cord blood erythrocytes. In the adult samples, HbF was detected in <5% of the circulating erythrocytes and gamma-globin gene expression in only 1.5% (3/206) of the reticulocytes. The average gamma-globin cDNA copy number in the minor population of gamma(+) adult reticulocytes was 468±198 copies, and the average beta-globin cDNA copy number in the beta(+) adult reticulocytes was 3869±3733 copies. Compared with the relatively monotonous patterns of gamma-globin gene and protein expression in cord and adult blood, we clearly detected an age-based fluctuation between those patterns in the infant blood samples. During the first three years of life, a gradual loss in the level of gamma-globin gene and protein expression was identified among the gamma(+)beta(+) reticulocytes and the HbF(+)HbA(+) erythrocytes. In addition, discrete populations of gamma(−)beta(+)reticulocytes and HbF(−)HbA(+) erythrocytes were detected. Rapid expansion of those gamma-silenced populations became apparent soon after birth. Within four months, the proportion of gamma-silenced cells eclipsed that of the gamma(+)beta(+) cells to become the predominant population. By three years after birth, the two cell populations essentially merged to become a single, gamma-silenced population similar to that found in adults. These data suggest two cellular mechanisms for gamma-globin silencing in humans: 1) a gradual loss in gamma-globin expression in the gamma(+)beta(+) cells beginning prior to delivery and continuing during infancy, and 2) replacement of the gamma(+)beta(+) cells with a population of gamma-silenced cells that rapidly accumulate after birth, possibly in response to the dramatic increase in oxygenation or other environmental changes.

Herpes Simplex Virus 1 Gene Expression Is Accelerated by Inhibitors of Histone Deacetylases in Rabbit Skin Cells Infected with a Mutant Carrying a cDNA Copy of the Infected-Cell Protein No. 0

ABSTRACT An earlier report showed that the expression of viral genes by a herpes simplex virus 1 mutant [HSV-1(vCPc0)] in which the wild-type, spliced gene encoding infected-cell protein no. 0 (ICP0) was replaced by a cDNA copy is dependent on both the cell type and multiplicity of infection. At low multiplicities of infection, viral gene expression in rabbit skin cells was delayed by many hours, although ultimately virus yield was comparable to that of the wild-type virus. This defect was rescued by replacement of the cDNA copy with the wild-type gene. To test the hypothesis that the delay reflected a dysfunction of ICP0 in altering the structure of host protein-viral DNA complexes, we examined the state of histone deacetylases (HDACs) (HDAC1, HDAC2, and HDAC3). We report the following. (i) HDAC1 and HDAC2, but not HDAC3, were modified in infected cells. The modification was mediated by the viral protein kinase US3 and occurred between 3 and 6 h after infection with wild-type virus but was delayed in rabbit skin cells infected with HSV-1(vCPc0) mutant, concordant with a delay in the expression of viral genes. (ii) Pretreatment of rabbit skin cells with inhibitors of HDAC activity (e.g., sodium butyrate, Helminthosporium carbonum toxin, or trichostatin A) accelerated the expression of HSV-1(vCPc0) but not that of wild-type virus. We conclude the following. (i) In the interval in which HSV-1(vCPc0) DNA is silent, its DNA is in chromatin-like structures amenable to modification by inhibitors of histone deacetylases. (ii) Expression of wild-type virus genes in these cells precluded the formation of DNA-protein structures that would be affected by either the HDACs or their inhibitors. (iii) Since the defect in HSV-1(vCPc0) maps to ICP0, the results suggest that this protein initiates the process of divestiture of viral DNA from tight chromatin structures but could be replaced by other viral proteins in cells infected with a large number of virions.

An Early Regulatory Function Required in a Cell Type-Dependent Manner Is Expressed by the Genomic but Not the cDNA Copy of the Herpes Simplex Virus 1 Gene Encoding Infected Cell Protein 0

ABSTRACT The α0 genes of herpes simplex virus 1 (HSV-1) contain three exons. Earlier studies have shown that the substitution of genomic sequences with a cDNA copy does not alter the capacity of the virus to replicate or establish latent infection. Other studies have demonstrated that HSV-1 may express alternatively spliced forms of α0 transcripts. The studies reported here centered on a mutant HSV-1(vCPc0) strain in which the genomic copies of the α0 gene were replaced with cDNA copies. From our research, we report the following observations. (i) In contrast to events transpiring in cells infected with wild-type virus, the expression of HSV-1(vCPc0) genes was delayed or restricted to α genes for many hours in rabbit skin cells and to a lesser extent in HEp-2 cells but not in Vero cells. This delay in the expression of HSV-1(vCPc0) β or γ genes was also multiplicity of infection dependent. (ii) Exposure to MG132, a proteasomal inhibitor, before infection with wild-type virus had no significant effect on the accumulation of viral proteins in Vero cells and caused an only slight delay in viral gene expression in rabbit skin cells in a multiplicity of infection-dependent fashion. The drug had no effect when it was added to the medium 3 h after infection. (iii) Rabbit skin or HEp-2 cells exposed to MG132 3 h after infection with the HSV-1(vCPc0) mutant accumulated only α proteins. This restriction was cell type dependent but not multiplicity of infection dependent. (iv) Both the delay in the expression of β and γ genes and the effect of MG132 added to the medium 3 h after infection were rescued by restoration of the intron 1 sequences in the HSV-1(vCPc0) mutant. However, cells transduced by baculoviruses expressing intron 1 RNA did not complement the HSV-1(vCPc0) mutant, suggesting that the function of intron 1 is in cis rather than in trans. We came to the following conclusions as a result. (i) Post-α gene expression requires the involvement of the proteasomal pathway in a cell type-dependent manner. Consistent with this requirement, the proapoptotic functions of MG132 are blocked in cells infected before exposure to the drug but not after exposure. (ii) A function encoded by the α0 gene that is absent from the cDNA copy is required for viral gene expression in a cell type- and multiplicity of infection-dependent fashion. The absence of this master function delays but does not ultimately block viral gene expression in the cell lines tested here.

Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.