H in α-Zr and in zirconium hydrides: solubility, effect on dimensional changes, and the role of defects.

Structural, thermodynamic and elastic properties of the hydrogen-zirconium system including all major hydrides are studied from first principles. Interstitial hydrogen atoms occupy preferentially tetrahedral sites. The calculations show that a single vacancy in α-Zr can trap up to nine hydrogen atoms. Self-interstitial Zr atoms attract hydrogen to a lesser extent. Accumulation of hydrogen atoms near self-interstitials may become a nucleation site for hydrides. By including the temperature-dependent terms of the free energy based on ab initio calculations, hydrogen adsorption isotherms are computed and shown to be in good agreement with experimental data. The solubility of hydrogen decreases in Zr under compressive strain. The volume dependence on hydrogen concentration is similar for hydrogen in solution and in hydrides. The bulk modulus increases with hydrogen concentration from 96 to 132 GPa.

[Role of tyrosine kinases in tumor progression of the head and neck]. / Die Rolle von Tyrosinkinasen bei Krebserkrankungen des Kopf-Hals-Bereichs.

Receptor tyrosine kinases play a key role in intercellular communication to maintain cellular integrity in tissues. Genetic alterations in these receptor-enzyme complexes or in their downstream signalling pathways may alter cellular characteristics such as proliferation, adhesion, differentiation and apoptosis. This result explains the role of these receptor complexes in the pathogenesis of malignancies. This article resumes the molecular basis and grouping of this receptor family and discusses the role of receptor alterations in carcinogenesis. This will be exemplified using the fibroblast growth factor receptor (FGFR) subfamily as well as the significance of single nucleotide polymorphism in cancer progression. The authors show that the application of molecular therapy is very promising and should be taken into consideration in concepts of multimodal cancer therapy.

Summary of workshop 'Theory Meets Industry'-the impact of ab initio solid state calculations on industrial materials research.

A workshop, 'Theory Meets Industry', was held on 12-14 June 2007 in Vienna, Austria, attended by a well balanced number of academic and industrial scientists from America, Europe, and Japan. The focus was on advances in ab initio solid state calculations and their practical use in industry. The theoretical papers addressed three dominant themes, namely (i) more accurate total energies and electronic excitations, (ii) more complex systems, and (iii) more diverse and accurate materials properties. Hybrid functionals give some improvements in energies, but encounter difficulties for metallic systems. Quantum Monte Carlo methods are progressing, but no clear breakthrough is on the horizon. Progress in order-N methods is steady, as is the case for efficient methods for exploring complex energy hypersurfaces and large numbers of structural configurations. The industrial applications were dominated by materials issues in energy conversion systems, the quest for hydrogen storage materials, improvements of electronic and optical properties of microelectronic and display materials, and the simulation of reactions on heterogeneous catalysts. The workshop is a clear testimony that ab initio computations have become an industrial practice with increasingly recognized impact.

Influence of cardiopulmonary resuscitation on levels of tumour markers.

Tumour markers (TM), including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen (CA) 15-3 and prostate-specific antigen (PSA), are serum markers for malignant diseases. Previous studies investigating the effect of acute and chronic inflammation, cardiopulmonary bypass surgery and cardiopulmonary resuscitation (CPR) on levels of TM showed conflicting results. Cardiopulmonary resuscitation (CPR) may result in a profound inflammatory response, and is frequently associated with severe tissue hypoperfusion. The present study investigated whether AFP, CEA, CA 15-3 and PSA are influenced by CPR. Alpha-fetoprotein (AFP), CEA, CA 15-3 and PSA (only in male patients) were assessed immediately after hospital admission, 6 h, 12 h and 2 days after prolonged CPR in eight male and 12 female patients. Serum levels of AFP, CEA, CA 15-3 did not change significantly after CPR. Prostate-specific antigen (PSA) levels increased significantly with a highest level in the study period 48 h after CPR (3.3 +/- 3.1 and 28.3 +/- 30.5 ng/mL for baseline and 48 h levels, respectively; P < 0.001). Alpha-fetoprotein (AFP), CEA, CA 15-3 and PSA (in men) values above the normal range were observed in 0%, 13.8%, 3.8% and 46.9% of all measurements respectively. At least one value above the normal range were observed in 0%, 20%, 5% and 75% of all patients for AFP, CEA, CA 15-3 and PSA (in men) respectively. Baseline values of AFP, CEA, CA 15-3 and PSA (in men) were above the normal range in 0%, 15%, 5% and 10% of all patients respectively. Levels for all markers did not differ significantly between survivors and non-survivors. In conclusion, prolonged CPR does not influence AFP, CEA, CA 15-3 serum levels, but is frequently associated with increases of PSA. Thus, in contrast to PSA, interpretation of AFP, CEA, CA 15-3 serum levels is not influenced by recent CPR.

Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus.

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.

The viral protein 3CD induces an equilibrium between the viral protein and RNA synthesis in a cell-free system for poliovirus replication.

In a cell-free system derived from uninfected HeLa cells and programmed with poliovirus RNA, an unbalance between the different replication steps is observed. After programming, the vRNA is exclusively used as a template for viral translation. It takes hours before there is a switch from protein synthesis to RNA replication. This is probably the reason for the inefficient production of infectious virus (compared to the synthesis in infected cells). If, however, the cell-free system is programmed with vRNA and with a mRNA coding for the viral protein 3CD, an increase in vRNA synthesis is found early post-programming, resulting in a better balance of protein synthesis and RNA synthesis and an increased virus yield of at least 2 log10. These data show that a balance between translation, RNA replication and packaging is required to allow efficient viral proliferation. The virus yield could be increased by a further log10 by the addition of pirodavir (a capsid stabiliser) and 10% of rabbit reticulocyte lysate to the cell-free system.

Viruses for the treatment of malignant glioma.

Viruses have been considered for use as therapeutic agents against cancer, and malignant glioma in particular. Oncolytic viruses were designed to target malignant cells supporting efficient virus replication, or rendered vulnerable to viral destruction due to tumor-specific defects in their defense against virus infection. Other than conventional cancer chemotherapy, viral antineoplastic agents require complex interactions with the host organism to reach their target and to unleash their oncolytic activity. Recent progress in the design and therapeutic application of oncolytic viruses carries the promise to make these agents available for treatment of malignant glioma.

Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2A(pro).

We have previously shown that the RNA polymerase 3D(pol) of human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969-10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5' end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3D(pol) (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359-10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [cre(2A)], located in the coding sequence of 2A(pro), serves as the primary template for HRV2 3D(pol) in the uridylylation of HRV2 VPg, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CD(pro). Our analyses suggest that HRV2 3D(pol) uses a "slide-back" mechanism during synthesis of the VPg-linked precursors. The corresponding cis- replicating RNA elements in the 2C(ATPase) coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) can be functionally exchanged in the assay with cre(2A) of HRV2. Mutations of either the first or the second A in the conserved A(1)A(2)A(3)CA sequence in the loop of HRV2 cre(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro VPg uridylylation reaction.

Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: purification and enzymatic analysis of the RNA-dependent RNA polymerase 3D(pol).

The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol). Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.

Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm].

We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G2-G6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5' upstream region.

The poliovirus receptor CD155 mediates cell-to-matrix contacts by specifically binding to vitronectin.

The human receptor for poliovirus (CD155) is an immunoglobulin-like molecule with unknown normal function(s). Here we provide evidence that CD155 binds specifically to vitronectin with a dissociation constant (K(d)) of 72 nM as determined by surface plasmon resonance. Based on sequence homology to the CD155 gene, three poliovirus receptor-related genes (PRR1, PRR2, and PRR3) were cloned recently. PRR proteins were reported by others to mediate homophilic cell adhesion. Neither PRR1 nor PRR2 binds poliovirus and it is assumed that their physiological functions differ from that of CD155. Indeed, mPRR2 was found to bind to vitronectin only weakly, while its self-adhesion activity is characterized by a K(d) of 310 nM. Moreover, there is no evidence for CD155 self-adhesion. Both CD155 and vitronectin colocalize to follicular dendritic cells and B cells inside the germinal centers of secondary lymphoid tissue (tonsils)-an observation suggesting that the CD155/vitronectin interaction is required for the establishment of a proper immune response in this particular context.

Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac.

The piggyBac transposable element was successfully used for stable genetic transformation of the housefly Musca domestica. The construct contains the EGFP marker under the control of Pax-6 binding sites, which can drive eye-specific expression in insect species as distantly related as Drosophila melanogaster and Tribolium castaneum [Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999) Nature 402: 370-371]. We obtained seven independent integration events among 41 fertile G0 Musca flies. Most of the transformed lines contained two or more chromosomal insertions of the EGFP marker which were stably inherited over more than 15 generations. piggyBac-mediated transposition was verified by identifying the characteristic TTAA duplication at the insertion sites. This first report of stable transmission of a genetic marker in Musca confirms the use of this vector-marker system for effective gene transfer in a broad range of insect species.

Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus.

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging to Flaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Deltacore-R-Luc-[PV]IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.

Polypeptide p41 of a Norwalk-like virus is a nucleic acid-independent nucleoside triphosphatase.

Southampton virus (SHV) is a member of the Norwalk-like viruses (NLVs), one of four genera of the family Caliciviridae. The genome of SHV contains three open reading frames (ORFs). ORF 1 encodes a polyprotein that is autocatalytically processed into six proteins, one of which is p41. p41 shares sequence motifs with protein 2C of picornaviruses and superfamily 3 helicases. We have expressed p41 of SHV in bacteria. Purified p41 exhibited nucleoside triphosphate (NTP)-binding and NTP hydrolysis activities. The NTPase activity was not stimulated by single-stranded nucleic acids. SHV p41 had no detectable helicase activity. Protein sequence comparison between the consensus sequences of NLV p41 and enterovirus protein 2C revealed regions of high similarity. According to secondary structure prediction, the conserved regions were located within a putative central domain of alpha helices and beta strands. This study reveals for the first time an NTPase activity associated with a calicivirus-encoded protein. Based on enzymatic properties and sequence information, a functional relationship between NLV p41 and enterovirus 2C is discussed in regard to the role of 2C-like proteins in virus replication.

Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1.

Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.

Will the polio niche remain vacant?

C-Cluster enteroviruses (C-CEVs), consisting of Coxsackie A viruses (C-CAV1, 11, 13, 15, 17, 18, 19, 20, 21, 22, 24, 24v) and polioviruses (PV1, 2, 3), have been grouped together in relation to their genomic sequences. On the basis of disease syndromes caused in humans, however, C-CAVs and PVs are vastly different: the former cause respiratory disease, just like the major receptor group rhinoviruses (magHRV), whereas PVs, on invasion of the CNS, can cause poliomyelitis. It is assumed that the difference in pathogenesis of C-CEVs is governed predominantly by cellular receptor specificity. C-CAVs use ICAM-1, just like magHRV, whereas PVs uniquely use CD155. Both ICAM-1 and CD155 are Ig-like molecules. Remarkably, based on a phylogenetic analysis of non-structural proteins, CAV 11, 13, 17 and 18 are interleaved with, rather than separated from, the three PV serotypes, e.g. PV1 is more closely related to CAV18 that to PV2. This observation suggests that PVs may have emerged from a pool of C-CAVs by evolving a unique receptor specificity. We have been studying virion structure, virion/receptor interactions, genetics, and the molecular biology of C-CEVs with the objective of identifying the molecular basis of phenotypic diversity of these viruses. Of particular interest is the prospect that C-CEVs can be genetically manipulated to switch their receptor affinity: from CD155 to ICAM-1 for PVs, or from ICAM-1 to CD155 for C-CAVs. We propose a hypothesis that in a world free of poliovirus and anti-poliovirus neutralizing antibodies C-CAVs would be given a greater chance to switch receptor specificity from ICAM-1 to CD155 and thus, to evolve gradually into a new polio-like virus.

The antiviral compound 5-(3,4-dichlorophenyl) methylhydantoin inhibits the post-synthetic cleavages and the assembly of poliovirus in a cell-free system.

The mode of action of the antiviral drug 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin) was studied in a cell-free system allowing de novo synthesis of poliovirus. This cell-free system, which is programmed with viral RNA, is suitable for the study of the late stages of poliovirus replication and, thus, for a study of antiviral compounds acting on these late stages. It was shown that, apart from the known inhibition of the assembly of poliovirus, hydantoin also blocks post-synthetic cleavages of poliovirus proteins. Our data demonstrate that the cell-free system is a sensitive tool to study the mode of action of antiviral compounds.

Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg.

The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation.

In addition to highly conserved stem-loop structures located in the 5'- and 3'-nontranslated regions, genome replication of picornaviruses requires cis-acting RNA elements located in the coding region (termed cre) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941-1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). cre elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the cre element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to the cre element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3D(pol) in a reaction that is greatly stimulated by 3CD(pro) (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359-10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conserved AAACA sequence in the loop of PV-cre(2C) eliminated the ability of the cre RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; AAACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild-type sequence. The effect of substitution of the third A (A4474G; AAACA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-cre(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.