Differential requirements for baculovirus late expression factor genes in two cell lines.

A plasmid library of 18 late expression factor (LEF) genes (LEF library) from the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) supports transient expression from a late viral promoter in the SF-21 cell line, derived from Spodoptera frugiperda. We found, however, that this LEF library was unable to support expression from the same promoter in the TN-368 cell line, derived from Trichoplusia ni, which is also permissive for AcMNPV replication. To identify the additional factor(s) required for expression in TN-368 cells, we cotransfected the LEF library with clones representing portions of the AcMNPV genome not represented in the LEF library. A single additional gene was identified; this gene corresponded to ORF70 of the complete AcMNPV sequence and potentially encodes a 34-kDa cysteine-rich polypeptide. Because of its differential effect on late gene expression in the two cell lines, we renamed ORF70 hcf-1 (for host cell-specific factor 1). hcf-1 was involved in expression from reporter plasmids under late and very late but not early promoter control, indicating that it was also a LEF gene. Plasmid DNA replication assays indicated that HCF-1 was involved in virus origin-specific DNA replication in TN-368 cells. Three LEF genes, ie-2, lef-7, and p35, required for optimal virus origin-specific plasmid DNA replication or stability in SF-21 cells had little or no influence in TN-368 cells. Thus, as determined by transient-expression assays, cell line-specific and potentially host-specific factors are required for origin-specific DNA replication or stability.     

Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.     

HCF-1 amino- and carboxy-terminal subunit association through two separate sets of interaction modules: involvement of fibronectin type 3 repeats

When herpes simplex virus infects permissive cells, the viral regulatory protein VP16 forms a specific complex with HCF-1, a preexisting nuclear protein involved in cell proliferation. The majority of HCF-1 in the cell is a complex of associated amino (HCF-1(N))- and carboxy (HCF-1(C))-terminal subunits that result from an unusual proteolytic processing of a large precursor polypeptide. Here, we have characterized the structure and function of sequences required for HCF-1(N) and HCF-1(C) subunit association. HCF-1 contains two matched pairs of self-association sequences called SAS1 and SAS2. One of these matched association sequences, SAS1, consists of a short 43-amino-acid region of the HCF-1(N) subunit, which associates with a carboxy-terminal region of the HCF-1(C) subunit that is composed of a tandem pair of fibronectin type 3 repeats, a structural motif known to promote protein-protein interactions. Unexpectedly, the related protein HCF-2, which is not proteolyzed, also contains a functional SAS1 association element, suggesting that this element does not function solely to maintain HCF-1(N) and HCF-1(C) subunit association. HCF-1(N) subunits do not possess a nuclear localization signal. We show that, owing to a carboxy-terminal HCF-1 nuclear localization signal, HCF-1(C) subunits can recruit HCF-1(N) subunits to the nucleus.     

Functional cross-talk of HIV-1 Tat with p53 through its C-terminal domain

Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.     

A novel function for human factor C1 (HCF-1), a host protein required for herpes simplex virus infection,in pre-mRNA splicing

Human factor C1 (HCF-1) is needed for the expression of herpes simplex virus 1 (HSV-1) immediate-early genes in infected mammalian cells. Here, we provide evidence that HCF-1 is required for spliceosome assembly and splicing in mammalian nuclear extracts. HCF-1 interacts with complexes containing splicing snRNPs in uninfected mammalian cells and is a stable component of the spliceosome complex. We show that a missense mutation in HCF-1 in the BHK21 hamster cell line tsBN67, at the non-permissive temperature, inhibits the protein's interaction with U1 and U5 splicing snRNPs, causes inefficient spliceosome assembly and inhibits splicing. Transient expression of wild-type HCF-1 in tsBN67 cells restores splicing at the non-permissive temperature. The inhibition of splicing in tsBN67 cells correlates with the temperature-sensitive cell cycle arrest phenotype, suggesting that HCF-1-dependent splicing events may be required for cell cycle progression.     

Impact of the interaction between herpes simplex virus type 1 regulatory protein ICP0 and ubiquitin-specific protease USP7 on activation of adeno-associated virus type 2 rep gene expression

Expression of the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 in transfected cells reactivates rep gene expression from integrated adeno-associated virus (AAV) type 2 genomes via a mechanism that requires both its RING finger and USP7 interaction domains. In this study, we found that the rep reactivation defect of USP7-binding-negative ICP0 mutants can be overcome by further deletion of sequences in the C-terminal domain of ICP0, indicating that binding of USP7 to ICP0 is not directly required. Unlike the case in transfected cells, only the RING finger domain of ICP0 was essential for rep gene reactivation during HSV-1 infection. However, mutants unable to bind to USP7 activate HSV-1 gene expression and reactivate rep gene expression with reduced efficiencies. These results further elucidate the role of ICP0 as a helper factor for AAV replication and illustrate that care is required when extrapolating from the properties of ICP0 in transfection assays to events occurring during HSV-1 infection.     

Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65.

A transient expression system was developed which results in efficient synthesis of the regulatory protein Vmw65 of herpes simplex virus type 1 in eucaryotic cells. The gene for Vmw65 was linked to the cytomegalovirus immediate-early (IE) promoter-enhancer region in a plasmid containing the simian virus 40 origin of replication. When transfected into COS cells, Vmw65 was expressed from this vector in 25 to 50% of the cells, with total levels of the protein approaching 20% of those observed in infected cells. Vmw65 expressed in this system is functional for specific DNA-binding complex formation with the host cell octamer-binding protein TRF and for transactivation of IE gene expression. We therefore produced a series of carboxy-terminal truncated forms of Vmw65 to examine the structural requirements of the protein for these activities. Deletion of the acidic carboxy-terminal 56 amino acids had no effect on DNA-binding complex formation but completely abolished the ability to transactivate. Amino acids between residues 434 and 453, a region which exhibits a high negative charge, were critical for IE transactivation. In contrast, the requirements for complex formation are located entirely within the N-terminal 403 amino acids, and our results indicate a requirement for this activity for residues between 316 and 403. Together with our previous work, the results presented here indicate that recruitment of TRF into a specific DNA-binding complex on IE consensus signals is required but not sufficient for functional IE transactivation by Vmw65.