Subgenomic Clones Research Articles

Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30.

BackgroundMuscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood.MethodsThe FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus.ResultsThe genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D′ sequence at the 3′ ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains revealed that FZ91-30 had five mutations; two in the unique region of the VP1 protein (VP1u) and three in VP3. Sequence alignment of the Rep1 proteins revealed two amino acid alterations for FZ91-30, both of which were conserved for two pathogenic strains YY and P. Transfection of the plasmid pFZ in 11-day-old embryonated goose eggs resulted in generation of infectious virus with similar biological properties as compared with the parental strain.ConclusionsThe amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV.

Development of a subgenomic clone system for Kyasanur Forest disease virus

Emerging tropical viruses pose an increasing threat to public health because social, economic and environmental factors such as global trade and deforestation allow for their migration into previously unexposed populations and ecological niches. Among such viruses, Kyasanur Forest disease virus (KFDV) deserves particular recognition because it causes hemorrhagic fever. This work describes the completion of an antiviral testing platform (subgenomic system) for KFDV that could be used to quickly and safely screen compounds capable of inhibiting KFDV replication without the requirement for high containment, as the structural genes have been replaced with a luciferase reporter gene precluding the generation of infectious particles. The coordination of KFDV kinetics with the replication characteristics of the subgenomic system has provided additional insight into the timing of flavivirus replication events, as the genetically engineered KFDV genome began replication as early as 2h post cellular entry. Possession of such antiviral testing platforms by public health agencies should accelerate the testing of antiviral drugs against emerging or recently emerged viruses mitigating the effects of their disease and transmission.

Impaired antiviral activity of interferon alpha against hepatitis C virus 2a in Huh-7 cells with a defective Jak-Stat pathway

BackgroundThe sustained virological response to interferon-alpha (IFN-α) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-α responsiveness between HCV 1 and other genotypes have not been elucidated.ResultsVirus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-α effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-α against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-α induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones.ConclusionsThe cellular Jak-Stat pathway is critical for a successful IFN-α antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.

Small Molecule Inhibitors of a Glycoside Hydrolase Attenuate Inducible AmpC-mediated β-Lactam Resistance

The increasing spread of plasmid-borne ampC-ampR operons is of considerable medical importance, since the AmpC beta-lactamases they encode confer high level resistance to many third generation cephalosporins. Induction of AmpC beta-lactamase from endogenous or plasmid-borne ampC-ampR operons is mediated by a catabolic inducer molecule, 1,6-anhydro-N-acetylmuramic acid (MurNAc) tripeptide, an intermediate of the cell wall recycling pathway derived from the peptidoglycan. Here we describe a strategy for attenuating the antibiotic resistance associated with the ampC-ampR operon by blocking the formation of the inducer molecule using small molecule inhibitors of NagZ, the glycoside hydrolase catalyzing the formation of this inducer molecule. The structure of the NagZ-inhibitor complex provides insight into the molecular basis for inhibition and enables the development of inhibitors with 100-fold selectivity for NagZ over functionally related human enzymes. These PUGNAc-derived inhibitors reduce the minimal inhibitory concentration (MIC) values for several clinically relevant cephalosporins in both wild-type and AmpC-hyperproducing strains lacking functional AmpD.

High Frequency of DefectivevpuCompared withtatandrevGenes in Brain from Patients with HIV Type 1-Associated Dementia

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated dementia (HAD) and other neurological disorders. The role of HIV regulatory and accessory proteins in the pathogenesis of these disorders is unclear. Here we analyzed sequences of tat, rev, and vpu genes in 55 subgenomic clones previously shown to encode functional env genes from brain and lymphoid tissues of four AIDS patients with HAD. Phylogenetic analysis showed distinct compartmentalization of tat, rev, and vpu genes in brain versus lymphoid tissues. Nine of 19 vpu sequences from brain of two patients had premature stop codons at positions between amino acids 2 and 30, compared with 0 of 8 from lymphoid tissues. Tat sequences from brain (n = 8 of 8) but not lymphoid (n = 0 of 6) tissue from one patient had a 35 amino acid truncation at the C-terminus. Rev sequences from the brain of one patient (n = 6 of 8) had a 5 amino acid truncation. These results demonstrate a high frequency of defective vpu compared with tat and rev genes in brain from HAD patients, and identify sequence variants of these regulatory/accessory genes that may influence the pathogenesis of HIV-associated neurological disease.

990 Hepatitis C virus genomic and sub-genomic clones activate interferon-stimulated response element in HUH-7 cells

990 Hepatitis C virus genomic and sub-genomic clones activate interferon-stimulated response element in HUH-7 cells

PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone.

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.

Restriction Fragment Length Polymorphism and Random Amplified Polymorphic DNA Analysis of Chickpea Accessions

Genetic diversity analysis was carried out in chickpea accessions using restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. RFLP analysis using 26 Pst I sub-genomic clones on ten chickpea accessions in 130 probe-enzyme combinations detected polymorphism with only two clones. Pst I clones, CG 141 detected polymorphism in ICC 4918 and Pusa 209 while CG 500 detected polymorphism in Pusa 261, ILC 26 and in ILC 13326. These clones detected very few polymorphic markers. Analysis using 10 Eco RI clones on twelve chickpea accessions have shown better hybridisation signal and one clone detected polymorphism in Pusa 256. RFLP analysis of both cultivated and wild Cicer species using heterologous DNA probe Cab3C revealed polymorphism only in wild Cicer species (Cicer reticulatum L., JM 2100). RAPD analysis of 13 chickpea accessions which includes mutants of C 235 and E100Y showed greater degree of polymorphism with 1 - 5 unique DNA bands for all the accessions. Phylogenetic analysis of the RAPD data helped to group the accessions. C 235 and its mutants were found to be closely grouped while E100Y and its mutant E100Ym grouped apart. Desi and kabuli chickpea accessions however, could not be separately grouped.

Characterization of chimeric full-length molecular clones of Aleutian mink disease parvovirus (ADV): identification of a determinant governing replication of ADV in cell culture

The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.

Regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus

The long terminal repeat (LTR) of a retrovirus contains sequence elements that constitute a promoter for controlling viral gene expression in infected cells. We have examined regulation of LTR-directed gene expression in feline immunodeficiency virus (FIV), a T-lymphocytopathic lentivirus associated with a fatal AIDS-like disease in domestic cats. Two independent virus isolates, designated FIV-Petaluma and FIV-PPR, have been molecularly cloned and show greater than 85% sequence homology. Both clones (termed pF34 and pPPR) produce infectious virus after transfection of permissive feline cells. Basal promoter activity of the LTRs was measured in various cell lines in transient expression assays using plasmids containing the viral LTR linked to the bacterial chloramphenicol acetyltransferase gene. Both LTRs were strong promoters in several cell lines, although in some cell lines the pF34 LTR had four- to fivefold higher basal activity than the pPPR LTR. FIV LTR mutations affecting the first AP4 site, AP1 site, ATF site, or NF-κB site resulted in decreased basal activity of the FIV promoter. Mutational analysis also revealed a negative regulatory element. In cotransfection experiments, both pF34 proviral DNA and pPPR proviral DNA appeared to transactivate either the pF34 LTR or the pPPR LTR; however, levels of transactivation were very low. Cotransfection of both LTRs with FIV subgenomic clones containing various viral open reading frames resulted in low level or no transactivation. The LTRs of both FIV clones responded to cell activation signals in human T-lymphoid cells (Jurkat) treated with phytohemagglutinin and phorbol-12-myristate-13-acetate. Promoter function of both FIV LTRs was also enhanced in cells treated with either forskolin, an inducer of intracellular cyclic-AMP (c-AMP), or dibutyryl c-AMP. Analysis of site-specific mutants showed that a potential AP1 site in the U3 domain of the LTR was required for T-cell activation responses mediated by protein kinase C, whereas a putative ATF site was the target for c-AMP-induced responses mediated by protein kinase A. These studies revealed that cellular transcription factors play a significant role in regulation of FIV gene expression.

Human papillomavirus type 16 and 18 gene expression in cervical neoplasias

Human papillomavirus (HPV) types 16 and 18 are strongly implicated in the generation of progressive cervical neoplasms. The viruses produce complex families of overlapping messenger RNAs that are linked to differentiation, making it necessary to analyze gene expression in the context of morphology. We have developed HPV type 16 and type 18 subgenomic clones from which 3H-labeled riboprobes specific to individual mRNA families can be generated in vitro. Using these probes for in situ hybridization, we examined serial sections of archival biopsy specimens of the spectrum of genital lesions. In low-grade squamous lesions, all viral open reading frames were expressed, and the most abundant transcription spanned the E4 and E5 open reading frames at the 3′ end of the E region. L region transcription coding for the capsid proteins was restricted to terminally differentiated keratinocytes. As the grade of neoplasia increased, cellular differentiation and overall viral transcription decreased and, with few exceptions, the L2 and L1 transcripts ceased to exist. The E6–E7 transforming region was invariably derepressed. Interestingly, the patterns of HPV-16 gene expression suggested the coexistence of episomal and integrated viral DNAs. In contrast, in HPV-18 lesions, all the viral template DNA appeared to have integrated. Integration was deduced to have occurred near the boundary of the E1 and E2 open reading frames. Viral transcription patterns were similar in carcinomas in situ and in invasive carcinomas, regardless of the histologic cell types or the associated virus types, consistent with the notion that additional host gene alterations were necessary for progression. On the basis of viral gene expression in vivo and the E6 promoter regulation previously characterized in vitro, we discuss a molecular mechanism for HPV-associated carcinogenesis.

The Tat protein of equine infectious anemia virus is encoded by at least three types of transcripts

Nucleotide sequence analysis of a cDNA library of EIAV-infected canine cells established a complex pattern of gene expression, characterized by alternatively spliced polycistronic transcripts. The EIAV tat gene product was shown to be encoded by at least three species of mRNA which differed in their ability to trans-activate the EIAV LTR upon expression in canine cells. The most active cDNA was monocistronic, consisting of three exons. The most abundant cDNA in the library contained four exons and was identical to a polycistronic transcript previously described (Noiman et al., 1990b) which contains open frames for Tat, putative Rev, and truncated transmembrane proteins. Products consistent in size with those predicted for these last two proteins could be detected in in vitro translation experiments. The third Tat message, another four-exon form, also potentially encodes an amino terminally truncated transmembrane protein. In vitro mutagenesis experiments and analysis of subgenomic and partial cDNA clones confirmed and extended previous findings that S1 sequences are essential for trans-activation and that Tat translation initiates at a non-AUG codon either in the full-length Tat message or in the genomic S1 open reading frame. The Tat protein (8 kDa) was detected in cells transfected with a Tat cDNA construct and in canine cells persistently infected with EIAV. The Tat activity of polycistronic mRNAs was lower than that of the monocistronic form, suggesting that the expression of the EIAV trans-activator may be subject to several levels of posttranscriptional control.

In vitro translation of hepatitis A virus subgenomic RNA transcripts

A subgenomic cDNA clone from hepatitis A virus strain HM175, composed of the last eight nucleotides of the 5' non-translated region and the first 2248 nucleotides of the coding sequence (P1 region), was inserted into a vector under the control of the T7 promoter. Restriction enzyme digestion at sites within the structural region and subsequent transcription in vitro yielded RNA products which were translated efficiently in rabbit reticulocyte lysates to produce proteins of the predicted sizes. The translation products were specifically precipitated with antipeptide antisera; these reactions were not affected by denaturation of the antigens by boiling in 1% SDS. The translated proteins were also precipitated by antivirion antisera, but recognition was totally abolished following denaturation. Thus antivirion antisera recognized conformation-dependent epitopes expressed on the translated products exclusively.

Structure of the Luteinizing Hormone Receptor Gene and Multiple Exons of the Coding Sequence*

The genomic structure of the LH receptor is important to our understanding of its expression mechanisms, functional domains, relationships with other hormone receptors, and evolution. We have isolated four overlapping cosmid clones and six subgenomic clones of the rat LH receptor gene. They span a total of 95.6 kilobases (kb) and extend from 23 kb upstream of the translation start site to 13 kb down-stream of the stop codon. In addition, part of the human LH receptor gene has been isolated. The coding region of the rat hormone receptor gene spans over 60 kb and consists of 11 exons and 10 introns. Southern blots hybridized with exon 1 and exon 11 probes as well as gene dose analyses demonstrate that a single copy gene encodes the rat LH receptor. Sequence comparison suggests that the porcine and human LH receptor genes have similar, if not identical, exon-intron structures. There is no consensus cAMP-responsive element within 600 basepairs up-stream of the translation start site in spite of the cAMP responsiveness of the LH receptor gene. There are, however, unconventional cAMP-responsive elements in the region: one which is identical, several which are homologous to the activating protein-2-binding elements, CCCCAGGC, and several sequences which are similar to the G-rich cAMP-responsive element found in P450c21, a steroid 21-hydroxylase. The first 10 exons encode the N-terminal half of the molecule, while exon 11 encodes the C-terminal half of the molecule. This last exon is the same in the rat and human genes. The DNA and amino acid sequences of the first 10 exons show significant similarities and reveal repetitive sequence motifs. They have similar sizes which occur in the range of 69 and 183 bases; 8 of them are from 69-81 bases. Despite these remarkable similarities, structural predictions of exons 1-10 show a diversity of structures. The N-terminal half of the LH receptor appears to have a folded structure, with frequent turns and an extensive surface area. Part of the surface is predicted to be covered by amphiphilic helices and beta structures, types of secondary structure frequently found at the interfaces between subunits or between 2 interacting molecules. The introns dividing these exons also share many similarities.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of Infectious Swine Vesicular Disease Virus from Cloned cDNA in Mammalian Cells

Full-length cDNA clones of the swine vesicular disease virus (SVDV) were constructed from subgenomic cDNA clones in the expression vector pSVL (pSVLS00). The direct transfection of mammalian cells with plasmid pSVLS00 results in the production of infectious virus. The recovered virus was neutralized completely by anti-SVDV guinea-pig serum, but did show a difference in plaque morphology from the parental virus.

Infectious in Vitro transcripts from a plum pox potyvirus cDNA clone

A full-length cDNA clone of the 9786 nt plum pox virus (PPV) RNA genome has been cloned downstream from a phage T7 RNA polymerase promoter. The RNAs synthesized by in vitro run-off transcription in the presence of the 5′ cap analog m 7GpppG were infectious in Nicotiana clevelandii plants. No infectivity was detected when the transcriptions were carried out in the absence of the cap analog. Inoculations of the local lesion host Chenopodium foetidum indicated that the infectivity of the synthetic transcripts was about 1% of that of the native viral RNA. An extra G present at the 5′ terminus of the transcripts was lost during their replication in plants, and the typical length distribution of the poly(A) tails was recovered. The viral RNA recovered from transcript-infected plants had approximately the same specific infectivity as native viral RNA. A G/A sequence heterogeneity found between different cDNA subgenomic clones was used to demonstrate that the infections were caused by the in vitro transcripts and were not the result of contamination.

Nucleotide sequence of a circular single-stranded DNA associated with coconut foliar decay virus

A circular single-stranded (ss) covalently closed (ccc) DNA associated with coconut foliar decay virus (CFDV) was purified, amplified by the polymerase chain reaction, and subcloned and its sequence established by analysis of overlapping subgenomic cDNA clones. The complete CFDV sequence comprised 1291 nucleotides and contained open reading frames for six proteins of molecular weight larger than 5 kDa. One of these (ORF1, 33.4 kDa) codes for a leucinerich protein with the nucleoside triphosphate-binding motif GXGKS and may possibly participate in virus replication. The putative viral protein encoded by ORF3 (6.4 kDa) is a positively charged arginine-rich protein with homology to the capsid protein of nuclear polyhedrosis virus, and may represent the CFDV coat protein. CFDV DNA can form a stable stem structure of 10 GC base pairs subtending a loop sequence which in one orientation closely resembles the motif TAATATTAC conserved in a similar structural arrangement within the geminivirus group. Otherwise no sequence homology to DNA-containing plant viruses of the gemini- or caulimovirus groups was found. CFDV therefore represents a new taxonomic group of plant viruses.

Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.

Sequences in the 5' Non-coding Region of Human Rhinovirus 14 RNA that Affect in vitro Translation

A subgenomic cDNA clone from human rhinovirus 14 (HRV-14), comprising the 5' non-coding region and the first 1182 nucleotides of the coding sequence, has been inserted into a vector under the control of the T7 promoter, and RNA was transcribed. Deletions in the 5' non-coding sequence modulated viral polyprotein synthesis significantly in a reticulocyte lysate system. Removal of the first 491 nucleotides had little effect, but deletion of a further 55 nucleotides (491 to 546) significantly increased the efficiency of the translation process. Further deletion to nucleotide 621 almost abolished translation, suggesting an essential role for the 546 to 621 nucleotide sequence. The efficiency of the translation process can also be influenced by the addition of ribosomal salt wash prepared from uninfected HeLa cells.