Dynamic nuclear localization of the baculovirus proteins IE2 and PE38 during the infection cycle: the promyelocytic leukemia protein colocalizes with IE2.

The early gene products IE2 and PE38 of Autographa californica multicapsid nuclear polyhedrosis virus localize to distinct nuclear domains after transient expression. Here, the nuclear localization pattern and the putative association with cellular proteins have been determined during virus infection to shed light on the functional significance of the nuclear domains. IE2 was always localized to distinct nuclear structures while PE38 was partly present in nuclear dots. Confocal imaging indicated colocalization of PE38 and IE2 to common domains, prominently at 2 h p.i. The nuclear dot localization of PE38 in infected cells was different from that in transfected cells. Hence, we have performed cotransfection experiments that suggested that a viral factor influences the nuclear distribution. Since the promyelocytic leukemia protein (PML) that localizes to distinct nuclear multiprotein complexes termed ND10/PODs in mammalian cells functions as a target for some immediate early viral proteins, we have investigated whether baculovirus proteins act similarly. Transiently expressed IE2 and PE38 were found to be associated with endogenous PML in the mammalian cell line BHK21. Infection with a recombinant virus that expresses the human pml gene in insect cells reveals IE2 and PML to be colocalized during the early phase of infection followed by a redistribution of both proteins. Taken together our results provide first evidence that the early baculovirus protein IE2 associates at least with one component of mammalian PODs during virus infection, suggesting that POD-like structures can be formed in insect cells.

Actin rearrangement-inducing factor of baculoviruses is tyrosine phosphorylated and colocalizes to F-actin at the plasma membrane.

In previous studies we have identified actin rearrangement-inducing factor 1 as an early gene product of Autographa californica multicapsid nuclear polyhedrosis virus that is involved in the remodeling of the actin cytoskeleton. We have constructed viral recombinants with a mutated Arif-1 open reading frame that confirm the causal link of Arif-1 expression and the actin rearrangement observed as accumulation of F-actin at the plasma membrane at 3 to 7 h postinfection. Infection with Arif mutant viruses leads to the loss of actin accumulation at the plasma membrane in TN-368 cells, although in the course of infection, early actin cables and nuclear F-actin are observed as in wild-type-infected cells. By immunofluorescence studies, we have demonstrated the localization of Arif-1 at the plasma membrane, and confocal imaging reveals the colocalization to F-actin. Accordingly, the approximately 47-kDa Arif-1 protein is observed exclusively in membrane fractions prepared at 4 to 48 h postinfection, with a decrease at 24 h postinfection. Phosphatase treatment suggests that Arif-1 is modified by phosphorylation. Antibodies against phosphotyrosine precipitate Arif-1 from membrane fractions, indicating that Arif-1 becomes tyrosine phosphorylated during the early and late phases of infection. In summary, our results indicate that functional Arif-1 is tyrosine phosphorylated and is located at the plasma membrane as a component of the actin rearrangement-inducing complex.

Mitochondrial DNA acts as potential promoter of the baculovirus RNA polymerase.

We have examined whether mitochondrial DNA could act as target of the RNA polymerase encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus, because the baculovirus late promoters and the control region of host mitochondrial DNA show a high degree of sequence similarity. In vitro transcription using mitochondrial DNA from Spodoptera frugiperda cells and nuclear extracts prepared from baculovirus infected cells demonstrates that mitochondrial DNA is recognized by the viral RNA polymerase. Transcriptional initiation occurs at TAAG sequences, although not all of the six TAAG motifs present in the mitochondrial DNA fragment are recognized. The TAAG motif in the control region served as weak transcriptional start site, but some of the TAAG motifs in the coding sequences of the adjacent tRNA and rRNA genes are recognized efficiently. The sequences flanking the TAAG motifs used as transcriptional start sites have a lower helix stability than the flanking sequences of the nonfunctional TAAG motifs. These results support the view that helix stability rather than sequence specificity is an important factor for recognition of TAAG motifs by the viral RNA polymerase.

The early baculovirus he65 promoter: On the mechanism of transcriptional activation by IE1.

We have initiated studies on the mechanism of early transcriptional activation of the early he65 promoter during infection with Autographa californica multicapsid nuclear polyhedrosis virus. This analysis is based on a comparison of the sequences required for he65 promoter activation with those sequences that support specific protein binding. The he65 promoter is located immediately downstream of the homologous region (hr) 4a. The sequences of hr4a are characterized by two imperfect palindromes of 24 bp. The results of transient expression assays indicate promoter activation in the presence of both the proximal palindrome and the known viral trans-regulator IE1. The results of mobility shift assays and DNaseI footprinting analyses reveal differences in specific protein binding at and close to the proximal palindrome depending on whether the nuclear protein extracts are prepared from uninfected or infected cells. The analysis of the protein binding complex at the proximal inverted repeat with extracts from infected cells suggests the involvement of both IE1 and IE0 as oligomers. The minimal protein binding sequences include the left half-site of the 24 bp repeat with 9 additional bp of the flanking sequences. The right half-site of the repeat also directs binding although with lower affinity as confirmed by phenanthroline-copper footprinting assays. Both half-sites of the repeat are thus essential for he65 promoter activation, suggesting that IE1 acts via cooperative binding. We conclude that the proximal inverted repeat is able to interact with both IE1 and IE0 although IE1 is sufficient for activation at least in transient expression assays.

In vitro transcription of pe38/polyhedrin hybrid promoters reveals sequences essential for recognition by the baculovirus-induced RNA polymerase and for the strength of very late viral promoters.

In vitro transcription was used to analyze the promoter specificity of the alpha-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5' untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.

Identification of the early actin-rearrangement-inducing factor gene, arif-1, from Autographa californica multicapsid nuclear polyhedrosis virus.

In response to Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) infection, a sequential rearrangement of the actin cytoskeleton occurs. Previous studies suggest that the penetration of nucleocapsids induces early actin cables followed by further changes of the actin cytoskeleton which depend on early viral gene expression. By transfection of a plasmid library into Trichoplusia ni TN-368 cells, we have identified an early viral gene, designated arif-1, that is able to induce actin rearrangement. The determination of the nucleotide sequence of arif-1 revealed one open reading frame potentially encoding a gene product of 45 kDa with no significant sequence homology to known proteins. After expression of arif-1 in transfected cells, the induced actin rearrangement, visualized by fluorescence microscopy, was comparable to the changes of the actin cytoskeleton at 3 to 7 h postinfection. These changes are based on early viral gene expression during the infection cycle. A causal link between arif-1 expression and actin rearrangement in infected cells is suggested by infection studies with the AcMNPV/Spodoptera frugiperda MNPV hybrid, which carries a deletion in the arif-1 gene. In transfection experiments the presence of the known viral transactivator IE1 was required in addition to ARIF-1 to induce actin rearrangement. IE1 was needed for promoter activation of the arif-1 gene, since arif-1 expression under the control of the early pe38 promoter was sufficient to induce actin rearrangement in transfected cells. Primer extension analyses showed that the arif-1 gene is transcribed only during the early phase of AcMNPV infection in T. ni TN-368 cells. There was a delay of about 1 h compared to ie1 transcription, which is in agreement with the assumption that IE1 transactivates the arif-1 promoter during infection.

Baculovirus transactivator IE1 is functional in mammalian cells.

IE1 of Autographa californica multicapsid nuclear polyhedrosis virus acts as a transactivator of several viral promoters in insect cells. Transient expression assays indicate that IE1 is involved in the activation of the early promoter he65 in both insect TN-368 and mammalian BHK-21 cell lines. IE1 activation of the he65 promoter was compared with IE1 activation of the early 39K promoter. In contrast to the he65 promoter, the 39K promoter was not inducible by IE1 in BHK-21 cells.

Baculovirus infection of Spodoptera exigua larvae: lacZ expression driven by promoters of early genes pe38 and me53 in larval tissue.

To follow the progression of infection of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) within tissues of its larval host, we have constructed AcMNPV recombinants carrying lacZ reporter genes under the control of the early virus promoters pe38 and me53, in addition to the authentic genes. The early promoter-lacZ gene cassettes were located upstream of the very late polyhedrin gene. In infected insect cell lines, pe38 transcription is initiated at an early promoter, while me53 transcripts start from both early and late sites. Transcriptional mapping of the duplicated me53 and pe38 promoters driving lacZ expression showed that they initiated at the same start sites as in the authentic genes. Expression of lacZ by these recombinants was compared to a recombinant driving beta-glucuronidase expression from the very late p10 promoter and lacZ expression from the constitutive heat shock protein 70 promoter of Drosophila melanogaster. After infection of Spodoptera exigua larvae with the different recombinants, we followed reporter gene expression and polyhedron formation in different tissues using immunohistochemistry and electron microscopy. LacZ expression, indicative of early viral transcriptional activity, was detected in nearly all larval tissues during the course of infection. In most tissues these early events were followed by pathophysiological changes associated with late and very late gene expression. However, p10 transcription and polyhedron formation were not observed in midgut goblet cells, Malpighian tubules and salivary glands. These results suggest that expression of early virus genes, such as me53 and pe38, is not restricted to larval tissues that are permissive for AcMNPV replication.

Expression of PE38 and IE2, viral members of the C3HC4 finger family, during baculovirus infection: PE38 and IE2 localize to distinct nuclear regions.

The pe38 gene of Autographa californica nuclear polyhedrosis virus represents one of the major early transcripts after viral infection. The function of the pe38 protein, which contains a C3HC4 zinc finger motif, is still not understood. We have raised polyclonal antiserum against the pe38 protein, PE38, produced in bacteria to investigate pe38 expression in the course of infection. A approximately 38-kDa polypeptide is first detectable at 2 h postinfection and decreases rapidly after 24 h. During the late phases of infection, a smaller protein of approximately 20 kDa which cross-reacts with the PE38-specific antiserum is visible at a constant level until 120 h postinfection. Since the pe38 gene shares a divergent promoter unit with the ie2 gene (formerly IEN), we have compared the expressions of the two genes. Polyclonal antibodies were raised against the bacterially expressed ie2 protein. The temporal expression pattern of the approximately 49-kDa ie2 protein is comparable to that of the approximately 38-kDa pe38 protein. Furthermore, both proteins are present in the nuclear fraction of A. californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells, but the approximately 38-kDa pe38 protein is also detectable in the cytoplasm while the smaller protein of approximately 20 kDa is exclusively present in the cytoplasmic fraction. Immunofluorescence analysis reveals that PE38 and IE2 localize to distinct regions within the nucleus mainly detected after transfection of pe38- and ie2-expressing constructs.

Deletion of the baculovirus ecdysteroid UDP-glucosyltransferase gene induces early degeneration of Malpighian tubules in infected insects.

Deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the Autographa californica nuclear polyhedrosis virus (AcNPV) genome increases the speed of killing of this virus (D. R. O'Reilly and L. K. Miller, Bio/Technology 9:1086-1089, 1991). Second-instar Spodoptera exigua larvae are killed more rapidly by the egt deletion mutant of AcNPV than by wild-type AcNPV. Unlike wild-type AcNPV-infected larvae, larvae infected with an egt deletion mutant molt and resume feeding as mock-infected larvae do. Wild-type AcNPV and egt deletion mutant recombinants marked with a lacZ gene were used to study their pathogenesis in insects. Histopathological investigation revealed that early degeneration of the Malpighian tubules, not the molting per se, may be the cause of this increased speed of killing by AcNPV.

Sequence and temporal appearance of the early transcribed baculovirus gene HE65.

We have identified the early transcribed HE65 gene by screening a cDNA library from polyadenylated RNA which was isolated at 1 h after infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus (AcNPV). Nucleotide sequencing analysis of the HE65-specific cDNA clone reveals one open reading frame of 1,662 nucleotides from which a protein of 65 kDa in size can be predicted. The HE65 gene is located downstream of the late transcribed p80 gene and upstream of the homologous region hr4left, which overlaps the 5' sequences of the HE65 gene. An HE65-specific transcript of about 1,800 nucleotides is detectable 2 h postinfection and remains stable during the late phases of infection. RNase protection and primer extension analyses demonstrate that transcripts from the early start site of HE65 continue to accumulate from 2 to 48 h postinfection, even in the presence of aphidicolin. Furthermore, transcriptional analysis of the HE65 gene indicates a lower intensity of early transcription in comparison with the very early transcribed genes IEN, PE38, and ME53.

Baculovirus gene ME53, which contains a putative zinc finger motif, is one of the major early-transcribed genes.

We have been investigating the first viral genes to be transcribed in Spodoptera frugiperda cells after the infection with Autographa california nuclear polyhedrosis virus in order to identify regulatory proteins required for the activation of early and/or late gene transcription. By screening a cDNA library from polyadenylated RNA transcribed at 1 h postinfection, we identified a gene which is designated ME53. This newly identified gene is located upstream of the late p74 gene and forms a divergent promoter unit with the immediate-early gene IE0. The determination of the nucleotide sequence of ME53 has revealed an open reading frame encoding a gene product of 53 kDa. Furthermore, the sequence data suggest a putative DNA binding motif, a zinc finger, whose functional significance has yet to be shown. Transcription of ME53 does not require previous viral protein synthesis, and during infection, the level of promoter activity seems to be independent of trans-acting viral factors, as suggested by transient expression studies.

Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein.

Regulatory elements interacting with DNA-binding proteins have been investigated in the promoter sequence of the early PE38 gene in the Autographa californica nuclear polyhedrosis virus (AcNPV). A GATA motif located 50 nucleotides upstream of the PE38 transcriptional start site is recognized differentially in the course of infection. As demonstrated by footprint and gel mobility shift assays, the GATA sequences TTATCT are protected by nuclear extracts from uninfected Spodoptera frugiperda cells and from S. frugiperda cells early postinfection (p.i.) but not by S. frugiperda cell extracts isolated 40 h p.i. We have compared the binding capacity of the insect GATA-like protein with that of the vertebrate GATA-1 factor identified as erythroid-specific factor. Our results indicate that a factor present in mouse erythroleukemia cells, presumably GATA-1, can bind to the insect GATA motif and vice versa. Evidence from transient expression studies suggests that the mutated GATA sequences do not influence PE38 promoter activity in cell culture.

Identification of the very early transcribed baculovirus gene PE-38.

We have started to identify early viral RNAs that are transcribed at 1 h after inoculation to investigate the mechanism involved in the regulation of early gene expression of Autographa californica nuclear polyhedrosis virus (AcNPV). Cloned viral DNA fragments were hybridized to Northern (RNA) blots of polyadenylated RNA isolated from Spodoptera frugiperda cells at 1, 2, and 6 h postinfection to localize very early transcripts. Subsequently we prepared a cDNA library of polyadenylated RNA transcribed at 1 h after inoculation to analyze the cDNA clones corresponding to the major early RNAs. We identified a gene located upstream of the immediate-early gene IE-N extending in the opposite direction. Because of the very early expression during AcNPV infection and the transient expression in uninfected cells, we conclude that we found an immediate-early gene, designated PE-38. The determination of the nucleotide sequence of PE-38 revealed one open reading frame potentially encoding a gene product of 38 kDa. Results of in vitro translation experiments suggest that a PE-38-specific polypeptide of approximately 38 kDa can be expressed. We have evidence from computer analyses that the predicted amino acid sequence includes two putative DNA-binding motifs, a zinc finger, and a leucine zipper.

The expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells.

This report presents a synopsis of recently published work in our laboratory on the molecular biology of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The following studies have been summarized. (1) On the mode of transcription of the AcNPV genome in insect cells. (2) Translation of proteins encoded in the 81.2 to 85.0 map unit segment of AcNPV. (3) Inserts of insect cell DNA in the AcNPV genome. (4) Expression of influenza (fowl plague) virus haemagglutinin in Spodoptera frugiperda insect cells, and successful immunization of chickens. (5) Synthesis of the influenza virus haemagglutinin in insect larvae by recombinant AcNPV. This insect virus system will continue to serve as a model for research on the molecular biology of insects. Moreover, the baculovirus system has been recognized as a very efficient and safe eukaryotic expression vector.

Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus.

Promoter inactivation by sequence-specific methylation was demonstrated by using a construct which contained the late E2A promoter of adenovirus type 2 (Ad2) DNA and the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as indicator. After the in vitro methylation of 5'-CCGG-3' sequences at positions -215, +6, and +24 relative to the cap site of the promoter, the construct was inactive upon transfection into mammalian cells. The same pAd2E2AL-CAT construct was active in the unmethylated form. Promoter inactivation could be overcome when the strong immediate early enhancer of human cytomegalovirus DNA, which lacked 5'-CCGG-3' sites, was inserted into the construct either in a position immediately antecedent to the promoter or in a location several thousand nucleotides remote from it. Reactivation of the 5'-CCGG-3' methylated pAd2E2AL-CAT construct entailed initiation of transcription at the authentic cap site of the late E2A promoter and maintenance of methylation at least during the duration of the transient expression experiment. Reactivation of the methylated late E2A promoter had also been demonstrated by the trans-activating 289 amino acid protein which was encoded in the E1A region of adenoviruses (B. Weisshaar et al., 1988, J. Mol. Biol. 202, 255-270). Thus there are several ways in which a methylated and silenced promoter can be reactivated in mammalian cells.