Soluble expression and complex formation of proteins required for HCMV DNA replication using the SFV expression system.

Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms-a precursor and a product-indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.

The acidic domain of pUL37x1 and gpUL37 plays a key role in transactivation of HCMV DNA replication gene promoter constructions.

Transient complementation of human cytomegalovirus (HCMV) oriLyt DNA replication in permissive human diploid cells expressing replication genes under native promoters requires its UL36-38 gene products. Two of the immediate early (IE) proteins encoded by this locus, pUL37x1 and, to a lesser extent, gpUL37, activated expression of HCMV early gene promoter constructions. The other IE protein encoded by the UL36-38 locus, pUL36, and the early product, pUL38, did not transactivate the HCMV early promoter constructions under similar conditions. The acidic domain, common to both pUL37x1 and gpUL37, is required for activation of HCMV early promoter constructions. Conversely, gpUL37 sequences downstream of amino acid 199 are not required for transactivation of viral early promoters. Taken together, these results suggest that the requirement for UL36-38 products for HCMV DNA replication results, at least in part, from the requirement of the transactivation of HCMV early DNA replication promoters by pUL37x1 and, to a lesser extent, by gpUL37 and that the acidic domain is critical for this activity.

Human cytomegalovirus oriLyt sequence requirements.

The mechanisms of action and regulation of the human cytomegalovirus (HCMV) lytic-phase DNA replicator, oriLyt, which spans more than 2 kbp in a structurally complex region near the middle of the unique long region (UL), are not understood. Because oriLyt is thought to be essential for promoting initiation of lytic DNA synthesis and may participate in regulating the switch between lytic and latent phases, we undertook a mutational study to better define its sequence requirements. Kanr gene cassette insertions located an oriLyt core region between nucleotides (nt) 91751 and 93299 that is necessary but not sufficient for replicator activity in transient assays. In contrast, insertions into auxiliary regions flanking either side of this core-also required for significant replicator activity-had little effect. To search for essential components within the core region, we made a series of overlapping, roughly 200-bp deletions, and qualitatively and quantitatively assessed the abilities of the resulting constructs to mediate replication. All but one of these deletions produced a significant (i.e., greater than twofold) loss of activity, arguing that sequences across this entire region contribute to replicator function. However, two particularly critical segments separated by a dispensable region, here called essential regions I and II, were identified. Within essential region I, which overlaps the previously identified early transcript SRT, two adjacent but nonoverlapping, roughly 200-bp deletions abolished detectable replication. No single element or motif from the left half of essential region I was found to be essential. Thus, essential region I probably promotes replication through the cooperation of multiple elements. However, four small deletions in the right half of essential region I, which included or lay adjacent to the conserved 31-nt oligopyrimidine tract (referred to as the Y block), abolished or virtually abolished oriLyt activity. Together, these results identify candidate oriLyt sequences within which molecular interactions essential for initiation of oriLyt-mediated DNA synthesis are likely to occur.

The variable 3' ends of a human cytomegalovirus oriLyt transcript (SRT) overlap an essential, conserved replicator element.

The genetically defined human cytomegalovirus (HCMV) lytic-phase replicator, oriLyt, comprises more than 2 kb in a structurally complex region that spans a variety of potential transcription control signals. Several transcripts originate within or cross oriLyt, and we are studying these oriLyt transcription units to determine whether they participate in initiating or regulating lytic-phase DNA synthesis. Results presented here establish the temporal accumulation and structure of the smallest replicator transcript, which we call SRT, and identify a single-sequence element essential to replicator function. SRT was detected as early as 2 h after HCMV infection of human fibroblast cells; transcript levels increased by 24 h and continued to increase thereafter. Consistent with its early appearance, treatment of HCMV-infected cells with the viral DNA polymerase inhibitor phosphonoformic acid had no effect on SRT accumulation; however, no SRT was detected in RNA preparations from cycloheximide-treated infected cells. Additional Northern (RNA) analysis localized the 0.2- to 0.25-kb SRT to an apparently noncoding segment near the center of the oriLyt core region. Reverse transcriptase PCR (rapid amplification of cDNA 5' ends [5'-RACE]) identified a single 5' end. In transient-transfection assays, the sequence immediately upstream of SRT functioned as a promoter responsive to HCMV infection when placed upstream of a reporter gene, suggesting that SRT is the product of a discrete transcription unit. RNA ligase-mediated 3'-RACE showed that SRT is not polyadenylated and has heterogeneous 3' ends within a roughly 45-nucleotide window overlapping an oligopyrimidine sequence having counterparts in the lytic-phase replicators of several herpesviruses. Mutation of the oligopyrimidine element showed that it is essential to oriLyt replicator function; it is the only essential single-sequence HCMV oriLyt replicator element described to date. Collectively, the location of SRT near the center of the oriLyt core region, its early expression, its overlapping relationship with a sequence element essential to replicator function, and its similarities to replicator transcripts in other systems suggest the possibility that SRT plays a role in initiating or regulating HCMV lytic-phase DNA synthesis.

Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection.

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J.A. Kerry, M.A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three- to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.

Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes.

As previously shown, 11 loci are required to complement human cytomegalovirus (HCMV) DNA replication in a transient-transfection assay (G. S. Pari and D. G. Anders, J. Virol. 67:6979-6988, 1993). Six of these loci encode known or candidate replication fork proteins, as judged by sequence and biochemical similarities to herpes simplex virus homologs of known function; three encode known immediate early regulatory proteins (UL36-38, IRS1/TRS1, and the major immediate early region spanning UL122-123); and two encode early, nucleus-localized proteins of unknown functions (UL84 and UL112-113). We speculated that proteins of the latter five loci might cooperate to promote and regulate expression of the six replication fork proteins. To test this hypothesis we made luciferase reporter plasmids for each of the replication fork gene promoters and measured their activation by the candidate effectors, expressed under the control of their respective native promoters, using a transient-cooperativity assay in which the candidate effectors were subtracted individually from a transfection mixture containing all five loci. The combination of UL36-38, UL112-113, IRS1, or TRS1 and the major immediate early region produced as much as 100-fold-higher expression than the major immediate early region alone; omitting any one of these four loci from complementing mixtures produced a significant reduction in expression. In contrast, omitting UL84 had insignificant (less than twofold), promoter-dependent effects on reporter activity, and these data do not implicate UL84 in regulating HCMV early-gene expression. Most of the effector interactions showed significant positive cooperativity, producing synergistic enhancement of expression. Similar responses to these effectors were observed for the each of the promoters controlling expression of replication fork proteins. However, subtracting UL112-113 had little if any effect on expression by the UL112-113 promoter or by the simian virus 40 promoter-enhancer under the same conditions. Several lines of evidence argue that the cooperative interactions observed in our transient-transfection assays are important to viral replication in permissive cells. Therefore, the data suggest a model in which coordinate expression of multiple essential replication proteins during permissive infection is vitally dependent upon the cooperative regulatory interactions of proteins encoded by multiple loci and thus have broad implications for our understanding of HCMV biology.

The human cytomegalovirus genes and proteins required for DNA synthesis.

Although the overall picture of HCMV DNA synthesis appears typical of the herpesviruses, some novel features are emerging. Six herpesvirus-group-common genes encode proteins that likely constitute the replication fork machinery, including a two-subunit DNA polymerase, a helicas-primase complex and a single-stranded DNA-binding protein. No homolog of the herpes simplex virus origin-binding helicase is evident, but at least one additional HCMV protein of unknown function, pUL84, appears to be required for initiation. Replication initiates within or near the large and structurally complex lytic-phase replicator, ori-Lyt, near the center of UL. Recent findings suggest that ori-Lyt-mediated initiation of DNA synthesis occurs through a mechanism distinct from that employed by herpes simplex virus.

Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication.

Recently we described the use of human cytomegalovirus (HCMV) cosmid clones in a cotransfection assay of HCMV oriLyt replication (G. S. Pari, M. A. Kacica, and D. G. Anders, J. Virol. 67:2575-2582, 1993). We have now used this assay to identify 11 distinct required loci encoding trans-acting factors sufficient for transient complementation of oriLyt-dependent DNA replication. This set includes all of the virus genes essential to initiate and perform DNA synthesis together with the virus genes required to express these replication functions from their native promoters. Six of the identified loci span open reading frames (ORFs) that encode homologs or probable homologs of herpes simplex virus type 1 replication genes, consistent with predictions based on sequence similarities and biochemical properties. These include the DNA polymerase UL54 and polymerase-associated protein UL44, the single-stranded-DNA-binding protein UL57, and proposed subunits of a helicase-primase complex, UL70, UL105, and UL101-102. Frameshift mutations in any one of these essential ORFs abrogated complementation of DNA replication. Three required loci, UL36-38, IRS1 (or TRS1), and IE1/IE2, encode known regulatory proteins. The remaining two loci span ORFs UL84 and UL112-113 and encode early temporal class nucleus-associated proteins of unknown function. Neither of these genes have been implicated previously in DNA replication or in regulating gene expression, nor have counterparts in herpes simplex virus type 1 or Epstein-Barr virus been described. The results presented here will facilitate investigation of the mechanisms and regulation of HCMV lytic-phase DNA replication.

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.

Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication.

We have localized a cis-acting sequence that promotes initiation of lytic-phase DNA replication (oriLyt) within the HindIII D fragment of the human cytomegalovirus (HCMV) AD169 genome and investigated its sequence requirements by testing the ability of plasmid constructs to mediate DNA replication in a transient transfection-plus-infection assay. Replication of plasmids containing HCMV oriLyt required at least the virus-specified DNA polymerase activity supplied by HCMV infection of transfected cells and was autonomous in that it did not result from recombination with the virus genome. Progeny molecules in the transient assay were high-molecular-weight tandem oligomers, which is consistent with predictions of a rolling-circle model. Experiments testing subclones of HindIII-D defined a core 2.4-kbp region containing elements required for oriLyt function that extended rightward from around 1.0 kbp upstream of UL57 near the middle of the long unique component of the virus genome. Sequences flanking this core also were needed for full activity. The defined region contains at least four clustered sets of repeated sequence elements identical to or candidate counterparts of elements present in the corresponding cytomegalovirus Colburn lytic-phase replication origin. These elements are novel in that they apparently do not correspond to previously characterized motifs. Also present are multiple copies of elements similar to known binding sites for the transcription factors ATF/CREB, MLTF/USF, and Sp1. Preliminary deletion analysis suggests that multiple components within the boundaries of oriLyt cooperate to enable initiation of HCMV lytic-phase DNA synthesis.

Multicomponent origin of cytomegalovirus lytic-phase DNA replication.

Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dbp. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A+T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.

Nucleotide sequence of a cytomegalovirus single-stranded DNA-binding protein gene: comparison with alpha- and gammaherpesvirus counterparts reveals conserved segments.

The genomic sequence encoding a cytomegalovirus strain Colburn homologue (DB129) of the herpes simplex virus major DNA-binding protein (ICP8) was determined. Multiple alignments of the deduced DB129 amino acid sequence and three alpha- and gammaherpesvirus homologues revealed that 56% of the amino acid residues identical in all four homologues are contained within 12 relatively conserved segments, which together constitute only 11.2% of the shortest aligned sequence. In light of published ICP8 deletion analyses, this alignment suggests conserved segments that may participate in forming DNA contacts. The identified conserved regions present interesting targets for site-directed mutagenesis in structure-function analyses.

Location, transcript analysis, and partial nucleotide sequence of the cytomegalovirus gene encoding an early DNA-binding protein with similarities to ICP8 of herpes simplex virus type 1.

The results presented here locate the gene encoding an early, nonvirion, single-stranded DNA-binding protein of human and simian strains of cytomegalovirus (CMV) [HCMV(Towne) DB140 and SCMV(Colburn) DB129, respectively] and provide additional evidence that this protein is the CMV homolog of the herpes simplex virus type 1 (HSV-1) major DNA-binding protein (ICP8), as proposed earlier (D. G. Anders, A. Irmiere, and W. Gibson, J. Virol. 58:253-262). The ICP8 gene was used as a probe in Southern analyses done at moderate stringency as an approach to locating similar sequences in the CMV genome. The BamHI K and EcoRI V fragments from the center of the long unique segment of HCMV(Towne) hybridized with the ICP8 probe and were in turn used to identify corresponding sequences in the EcoRI D fragment of SCMV(Colburn). RNA prepared from SCMV(Colburn)-infected cells directed the in vitro synthesis of DB129. If the RNA was first hybridized with the cloned 12.5-kilobase EcoRI D fragment, in vitro synthesis of DB129 was specifically inhibited. Additional hybrid-arrested in vitro translation experiments with subclones spanning the EcoRI D fragment demonstrated that the DB129 gene is located in the left half of that fragment, approximately bisected by a SalI site. RNA analyses identified 3.9-, 8.9-, and 10.0-kilobase RNA species expressed from this region. A partial nucleotide sequence of the Colburn region mapping within the boundaries of the 3.9-kilobase transcript, suspected to be the primary coding species, showed significant sequence similarity to the major DNA-binding protein gene homolog identified in B95-8 Epstein-Barr virus.

Immunological characterization of an early cytomegalovirus single-strand DNA-binding protein with similarities to the HSV major DNA-binding protein.

Monospecific polyclonal antisera were prepared against the 129-kDa, early, single-strand DNA-binding protein (DB129) of strain Colburn cytomegalovirus (CMV), and used to study its distribution in infected cells and its relatedness to a proposed human CMV (HCMV) counterpart (DB140). Indirect immunofluorescence of fixed, infected human fibroblasts showed DB129 to be localized within the intranuclear inclusions characteristic of replicating CMV. Treatment of infected cells with 50 to 100 micrograms phosphonoformic acid per milliliter resulted in the overproduction of DB129 and its accumulation within nuclei, both inside the inclusions and in surrounding areas of the nucleoplasm, whereas treatment with 500 micrograms/ml prevented inclusion formation, and DB129 was localized at discrete points throughout the infected-cell nuclei. The sera cross-reacted an estimated 10% with HCMV DB140 in an indirect immunoassay, and their use in immunofluorescence localized DB140 to the nuclear inclusions of HCMV-infected cells. Their immunological cross-reactivity, as well as their similar biochemical properties and intracellular distribution, support the likelihood that DB129 and DB140 are the protein products of homologous genes. The relationship of these proteins to the herpes simplex major DNA-binding protein is discussed.

Identification and characterization of a major early cytomegalovirus DNA-binding protein.

We characterized a DNA-binding protein with an approximate molecular weight of 129,000 (DB129) which is present in the nuclei of cytomegalovirus- (strain Colburn) infected cells, but not in virus particles. Results of two types of experiments demonstrated that DB129 is a member of the early class of herpesviral proteins. First, time course pulse-labeling experiments showed that its synthesis begins after that of the immediate-early protein IE94, but prior to the appearance of late viral proteins, and was reduced at late times. Second, in the presence of inhibitors of viral DNA replication, DB129 continued to be made and accumulated to elevated levels. A second set of experiments showed that DB129 bound to single-stranded DNA in vitro and was eluted by a NaCl gradient in two peaks, one at about 0.2 M and the second at about 0.6 M. A similar pattern of release was observed when infected-cell nuclei were serially extracted with increasing NaCl concentrations. In addition, treatment of nuclei with DNase I selectively released DB129, along with a small but significant fraction of another DNA-binding protein, DB51. These results suggest that DB129 is associated with DNA in vivo and that it interacts directly with single-stranded DNA. It was also shown that cells infected with human cytomegalovirus (strain Towne) contain a slightly larger counterpart to DB129, which was designated DB140. Similarities between these proteins and the major DNA-binding protein of herpes simplex virus are discussed.

Chemical cleavage of polyomavirus major structural protein VP1: identification of cleavage products and evidence that the receptor moiety resides in the carboxy-terminal region.

As a first step toward identifying the various functional regions of the polyomavirus major capsid protein VP1, we used recently developed methods for the chemical cleavage of proteins and the available polyomavirus sequence data to devise a scheme to produce large, identifiable peptides and generate a cleavage map of VP1. Formic acid (75%) was found to cleave VP1 at only two sites, producing three peptides of apparent molecular weights of 29,000, 16,000, and 2,000. The order of peptides in intact VP1 was determined by recleavage of partial products and was found to be 29,000, 16,000, and 2,000. Two-dimensional peptide mapping studies of 125I-labeled VP1 formic acid peptides established that the limit products of formic acid digestion contained mutually exclusive sets of labeled peptides when either trypsin or chymotrypsin was used and that together the formic acid peptides contained all of the 125I-labeled tryptic and chymotryptic peptides found in VP1. Iodosobenzoic acid (IBA) digestion produced four peptides separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 12,000, 8,000, 7,000, and 5,000. The approximate positions of the IBA peptides in the VP1 sequence were determined by cleavage of formic acid fragments with IBA. The number of peptides produced, their respective sizes, and their order in the intact VP1 molecule agree with predictions made from available sequence data, both for formic acid cleavage and IBA cleavage. In addition, the numbers of 125I-labeled tryptic peptides produced from digestion of VP1 formic acid peptides also agree with predictions made from the sequence information. These data establish with reasonable certainty that the peptides produced by formic acid cleavage and IBA cleavage of VP1 are indeed those predicted. Antibodies raised against spontaneously produced, previously undefined polypeptides resulting from degradation of VP1 reacted exclusively with the formic acid peptides derived from the C-terminal portion of VP1. These antibodies inhibited hemagglutination and neutralized polyomavirus virions. We interpret this to mean that at least some of the antigenic determinants of the receptor moiety reside in this portion of the VP1 sequence.

Comparison of nonphosphorylated and phosphorylated species of polyomavirus major capsid protein VP1 and identification of the major phosphorylation region.

The major virion protein of polyomavirus, VP1, consists of about six isoelectric species designated A through F. The minor species D, E, and F are phosphorylated and are thought to serve as viral receptors. We first wanted to distinguish whether all VP1 species are derived by post-translational modification from a common amino acid sequence or whether one or more of the species contain a region(s) of altered amino acid sequence resulting from alternate mRNA processing. We compared the VP1 species by detailed peptide mapping with several combinations of specific protease and radioisotopic labels. This approach enabled us to examine more than 80% of the predicted VP1 sequence, including the amino-and carboxy-termini. We found no evidence of sequence differences among any of the VP1 species. The specific incorporation of 32Pi was found to be the same for all of the phosphorylated species. Comparison of the phosphorylation sites of in vivo 32Pi-labeled D, E, and F by peptide mapping showed them to be identical. Each phosphorylated species contained a single major phosphopeptide and several minor phosphopeptides. The major phosphoamino acid, identified by acid hydrolysis, was phosphothreonine, with phosphoserine also present. By using chemical cleavage methods, we localized the major phosphorylation region to a central portion of the VP1 sequence. We discuss some features of this region and relate this information to functional implications of phosphorylation.

Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.

The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50.