Cell-free synthesis of polyoma virus capsid proteins VP1 and VP2.

Polyadenylated RNA isolated from the cytoplasm of mouse 3T6 cells 28 h after infection with polyoma virus has been isolated and translated in vitro. Polyoma capsid proteins VP1 and VP2 have been identified in the cell-free product by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide fingerprinting. Polyoma mRNA species have been isolated by preparative hybridization to purified viral DNA immobilized on cellulose nitrate filters and shown to code for both VP1 and VP2. These experiments establish conditions for the isolation of late polyoma mRNA and the cell-free synthesis of polyoma capsid proteins and indicate that the active mRNA species are at least partially virus coded.     

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.     

Characterization of the mRNA''s for the polyoma virus capsid proteins VP1, VP2, and VP3.

Polyadenylated cytoplasmic RNA from polyoma virus-infected cells can be translated in the wheat germ system to yield all there polyoma virus capsid proteins, VP1, VP2, and VP3. The translation products of RNA selected from total cytoplasmic RNA of infected cells by hybridization to polyoma virus DNA showed a high degree of enrichment for VP1, VP2, and VP3. The identity of the in vitro products with authentic virion proteins was established in two ways. First, tryptic peptide maps of the in vitro products were found to be essentially identical to those of their in vivo counterparts. Second, the mobilities of the in vitro products on two-dimensional gels were the same as those of viral proteins labeled in vivo. VP1, VP2, and vp3 were all labeled with [35S] formylmethionine when they were synthesized in the presence of [35S] formylmethionyl-tRNAfmet. We determined the sizes of the polyadenylated mRNA's for VP1, VP2, and VP3 by fractionation on gels. The sizes of the major mRNA species for the capsid proteins are as follows: VP2, 8.5 X 10(5) daltons; VP3, 7.4 X 10(5) daltons; and VP1, 4.6 X 10(5) daltons. We conclude that all three viral capsid proteins are synthesized independently in vitro, that all three viral capsid proteins are virally coded, and that each of the capsid proteins has a discrete mRNA.     

Polyoma virus has three late mRNA''s: one for each virion protein.

Polyoma virus mRNA, isolated from the cytoplasm of 3T6 cells late after infection and purified by hybridization to HpaII fragment 3 of polyoma virus DNA, was separated on 50% formamide-containing sucrose density gradients, and the fractionated RNA was recovered and translated in vitro. Analysis of the cell-free products showed that the minor virion protein VP3 was synthesized from an mRNA sedimenting at approximately 18S betweeen the 19S VP2 mRN and the 16S VP1 mRNA. Other experiments showed that the VP2 and VP3 can be labeled with formyl methionine from initiator tRNA. We conclude that there are three late polyoma virus mRNA's, each directing the synthesis of only one viral capsid protein.     

Polyoma Virus DNA: Complete Nucleotide Sequence of the Gene Which Codes for Polyoma Virus Capsid Protein VP1 and Overlaps the VP2/VP3 Genes

The nucleotide sequence of part of the late region of the polyoma virus genome was determined. It contains coding information for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. In the sequence with the same polarity as late mRNA's, all coding frames are blocked by termination codons in a region around 48 units on the physical map. This is the region where the N-terminus of VP1 and the C-termini of VP2 and VP3 have been located (T. Hunter and W. Gibson, J. Virol. 28:240-253, 1978; S. G. Siddell and A. E. Smith, J. Virol. 27:427-431, 1978; Smith et al., Cell 9:481-487, 1976). There are two long uninterrupted coding frames in the late region of polyoma virus DNA. One lies at the 5' end of the sequence and contains potential coding sequences for VP2 and VP3. The other contains 383 consecutive sense codons starting with the ATG at nucleotide position 1,218, extends from 47.5 to 25.8 units counterclockwise on the physical map, and is located where the VP1 gene has been mapped. The VP1 gene overlaps the genes for proteins VP2/VP3 by 32 nucleotides and uses a different coding frame. From the DNA sequence, the amino acid sequence of VP1 was predicted. The proposed VP1 sequence is in good agreement with other data, namely, with the partial N-terminal amino acid sequence and the total amino acid composition. The VP1 coding frame terminates with a TAA codon at 25.8 map units. This is followed by an AATAAA sequence, which may act as a processing signal for the viral late mRNA's. When both nucleotide and amino acid sequences are compared with their counterparts in the related simian virus 40, extensive homologies are found over the entire region of the two viral genomes. Maximum homology appears to occur in those regions which code for the C-termini of the VP1 proteins. The overlap region of VP1 with VP2/VP3 of polyoma virus is shorter by 90 nucleotides than is that of simian virus 40 and shows very limited homology with the simian virus 40 sequence. This leads to the suggestion that the overlap segments of both viruses have been freed from stringency imposed on drifting during evolution and that proteins VP2 and VP3 of polyoma virus may have been truncated by the appearance of a termination codon within the sequence.     

Topography of the three late mRNA''s of polyoma virus which encode the virion proteins.

The three cytoplasmic polyadenylated mRNA's which separately encode the three capsid proteins (VP1, VP2, and VP3) of polyoma virus were mapped on the viral genome by one- and two-dimensional gel electrophoreses of nuclease S1-resistant RNA-DNA hybrids. The mRNA's, which we designated mVP1, mVP2, and mVP3 to indicate the coding functions deduced from the cosedimentation of the RNAs and the messenger activities, comprise an overlapping set of 3'-coterminal molecules which also share a heterogeneous family of noncoding 5'-terminal regions (Flavell et al., Cell 16:357--371, 1979; Legon et al., Cell 16:373--388, 1979). The three species differ in the length of the 3' colinear coding region which is spliced to the 5' leader sequences. The common polyadenylated 3' end maps at map unit 25.3. The 5' ends of the colinear bodies of mVP1, mVP3, and mVP2 map at 48.5, 59.5, and 66.5 map units, respectively. An examination of the polyoma virus DNA sequence (Arrand et al., J. Virol. 33:606--618, 1980) in the vicinities of splicing sites approximated by the S1 gel mapping data for sequences common to the ends of known intervening sequences allowed prediction of the precise splice points in polyoma virus late mRNA's. In all three cases, the leader sequences are joined to the mRNA bodies at least 48 nucleotides before the translational initiation codon used in each particular messenger. The start signal which functions in each mRNA is the first AUG (or GUG) triplet after the splice junction.     

Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro.

The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5.     

Polyoma virus DNA: Sequence from the late region that specifies the leader sequence for late mRNA and codes for VP2, VP3, and the N-terminus of VP1.

The DNA sequence of part of the late region of the polyoma virus genome is presented. This sequence of 1,348 nucleotide pairs encompasses the leader region for late mRNA and the coding sequence for the two minor capsid proteins VP2 and VP3. The coding sequence for the N-terminus of the major capsid protein overlaps the C-terminus of VP2/VP3 by 32 nucleotide pairs. From the DNA sequence the sizes and sequences of VP2 and VP3 could be predicted. Potential splicing signals for the processing of late mRNA's could be identified. Comparisons are made between the sequence of polyoma virus DNA and corresponding regions of simian virus 40 DNA.     

Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40.

Numerous viral and cellular RNAs are polycistronic, including several of the late mRNA species encoded by simian virus 40 (SV40). The functionally bicistronic major late 16S and functionally tricistronic major late 19S mRNA species of SV40 contain the leader-encoded open reading frames (ORFs) LP1, located upstream of the sequence encoding the virion protein VP1, and LP1*, located upstream of the sequence encoding the virion proteins VP2 and VP3. To determine how these leader ORFs affect synthesis of the virion proteins, monkey cells were transfected with viral mutants in which either the leader-encoded translation initiation signal was mutated or the length and overlap of the leader ORF relative to the ORFs encoding the virion proteins were altered. The levels of initiation at and leaky scanning past each initiation signal were determined directly by quantitative analysis of the viral proteins synthesized in cells transfected with these mutants. Novel findings from these experiments included the following. (i) At least one-third of ribosomes bypass the leader-encoded translation initiation signal, GCCAUGG, on the SV40 major late 16S mRNA. (ii) At least 20% of ribosomes bypass even the consensus translation initiation signal, ACCAUGG, when it is situated 10 bases from the 5' end on the major late 16S mRNA. (iii)O The presence of the leader ORF on the bicistronic 16S mRNA species reduces VP1 synthesis threefold relative to synthesis from a similar RNA that lacks it. (iv) At least half and possibly all VP1 synthesized from the bicistronic 16S mRNA species is made by a leaky scanning mechanism. (v) LP1 and VP1 are synthesized from the bicistronic 16S mRNA species at approximately equal molar ratios. (vi) Approximately half of the VP1 synthesized in SV40-infected cells is synthesized from the minor, monocistronic 16S mRNA even though it accounts for only 20% of the 16S mRNA present. (vii) The presence and site of termination of translation of the leader ORF on the late 19S mRNAs affect the relative as well as absolute rates of synthesis of VP2 and VP3.     

Polyoma virus early and late mRNAs in productively infected mouse 3T6 cells.

We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The polyoma early mRNAs, which code for the three T antigens, have several 5' ends near 73 map units (m.u.). During the late phase of infection an additional 5' end is found near 71 m.u. All of the major early mRNAs have common 3' ends at 26.01 m.u. There is a minor species of early mRNA with a 3' end at 99.05 m.u. There are two proximal and two distal splice junctions in the early region which are used to generate three different spliced early mRNAs. There are three late mRNAs encoding the three virion proteins, VP1, VP2, and VP3. The late mRNAs have common 3' ends at 25.34 m.u. The late mRNAs have heterogeneous 5' leader sequences derived from the region between 65.53 and 68.42 m.u. The leader sequences are joined to the bodies of the messages coding for VP2, VP3, and VP1 at 66.59, 59.62, and 48.57 m.u., respectively. These results confirm and extend previous analyses of the fine structure of polyoma mRNAs.     

The 3' end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.     

Functional analysis of the triplex proteins (VP19C and VP23) of herpes simplex virus type 1

The triplex of herpesvirus capsids is a unique structural element. In herpes simplex virus type 1 (HSV-1), one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent VP5 molecules. The interaction between VP19C and VP23 was inferred by yeast cryoelectron microscopy studies and subsequently confirmed by the two-hybrid assay. In order to define the functional domains of VP19C and VP23, a Tn7-based transposon was used to randomly insert 15 bp into the coding regions of these two proteins. The mutants were initially screened for interaction in the yeast two-hybrid assay to identify the domains important for triplex formation. Using genetic complementation assays in HSV-1-infected cells, the domains of each protein required for virus replication were similarly uncovered. The same mutations that abolish interaction between these two proteins in the yeast two-hybrid assay similarly failed to complement the growth of the VP23- and VP19C-null mutant viruses in the genetic complementation assay. Some of these mutants were transferred into recombinant baculoviruses to analyze the effect of the mutations on herpesvirus capsid assembly in insect cells. The mutations that abolished the interaction in the yeast two-hybrid assay also abolished capsid assembly in insect cells. The outcome of these experiments showed that insertions in at least four regions and especially the amino terminus of VP23 abolished function, whereas the amino terminus of VP19C can tolerate transposon insertions. A novel finding of these studies was the ability to assemble herpesvirus capsids in insect cells using VP5 and VP19C that contained a histidine handle at their amino terminus.     

Mutations in VP1 of poliovirus specifically affect both encapsidation and release of viral RNA.

The phenotypic defects of two type 1 Mahoney poliovirus mutants, termed VP1-101 and VP1-102, were caused by two different small deletions in the region of the RNA genome encoding the amino terminus of the capsid protein VP1. This portion of VP1 was unresolved in the three-dimensional structure of the poliovirion, buried within the virion, and likely to interact with the viral RNA. Both VP1-101 and VP1-102 showed a diminished ability to enter CV1 but not HeLa cells; both mutants formed plaques on CV1 and HeLa cells that were smaller than wild type. Neither the rate of binding to cells nor the rate of subsequent receptor-dependent conformational change of the mutant poliovirions was affected. However, both mutants displayed delayed kinetics of RNA release compared with wild-type virus. One of the mutants, VP1-102, also displayed a defect in viral morphogenesis: 75S empty capsids formed normally, but 150S particles that contained RNA accumulated much more slowly. We suggest that the VP1-102 mutation affects RNA encapsidation as well as RNA release, whereas the VP1-101 mutation affects only RNA release. Therefore, RNA packaging and RNA release are genetically linked but can be mutated separately in different VP1 alleles, and both processes involve the amino terminus of VP1.     

Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.     

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Polyoma-induced stimulation of cellular RNA synthesis is paralleled by changed expression of the viral genome.

We studied synthesis of viral and cellular RNA in the presence and absence of 5-fluorodeoxyuridine (FdU, an inhibitor of DNA synthesis) during lytic infection with polyoma virus in confluent, primary mouse kidney cell cultures. In the presence of FdU, synthesis of early 19S polyoma mRNA and of polyoma tumor (T)-antigen, i.e. expression of the early viral gene, is rapidly followed by a mitogenic reaction of the host cell; it leads to an increase of 30 +/- 5% in cellular, mainly 28S and 18S rRNA, followed by activation of the cellular DNA-synthesizing apparatus. Polyoma-induced cellular RNA synthesis is paralleled by increased production of early 19S mRNA and begin of expression of the late viral genes, leading to synthesis of small amounts of late 19S and 16S mRNAs. Changed expression of the viral genome occurs in the absence of detectable synthesis of polyoma DNA I. Infection in the absence of FdU induces the same sequence of events; it is followed, however, by duplication of the mouse cell chromatin (S-phase) and production of progeny virus.