Role of early and late replication events in induction of apoptosis by baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) mutants that lack the apoptotic suppressor gene p35 cause apoptosis in Spodoptera frugiperda SF21 cells. To identify a viral signal(s) that induces programmed cell death, we first defined the timing of apoptotic events during infection. Activation of a P35-inhibitable caspase, intracellular fragmentation of host and AcMNPV DNA, and cell membrane blebbing coincided with the initiation of viral DNA synthesis between 9 and 12 h after infection and thus suggested that apoptotic signaling begins at or before this time. Virus entry was required since binding of budded virus to host cell receptors alone was insufficient to induce apoptosis. To therefore determine the contribution of early and late replication events to apoptotic signaling, we used the AcMNPV mutant ts8 with a temperature-sensitive lesion in the putative helicase gene p143. At the nonpermissive temperature at which viral DNA synthesis was conditionally blocked, ts8 caused extensive apoptosis of the SF21 cell line p3576D, which dominantly interferes with anti-apoptotic function of viral P35. Confirming that apoptosis can be induced in the absence of normal viral DNA synthesis, parental SF21 cells also underwent apoptosis when infected with a ts8 p35 deletion mutant at the nonpermissive temperature. However, maximum levels of ts8 p35 deletion mutant-induced apoptosis required a temperature-sensitive event(s) that included the initiation of viral DNA synthesis. Collectively, these data suggested that baculovirus-induced apoptosis can be triggered by distinct early (pre-DNA synthesis) and late replicative events, including viral DNA synthesis or late gene expression.     

The 35-kilodalton protein gene (p35) of Autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses.

Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinants were constructed to test the effectiveness of the AcMNPV 35-kilodalton protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of the AcMNPV apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in Spodoptera frugiperda (SF21) cell cultures. When placed under control of an early virus promoter, the bacterial neomycin resistance gene (neo) restored multiplication of AcMNPV in the same cells treated with concentrations of the antibiotic G418 that inhibited wild-type virus growth greater than 1000-fold. The selectivity of these dominant markers was compared by serial passage of recombinant virus mixtures. After four passages, the proportion of p35-containing virus increased as much as 2,000,000-fold relative to deletion mutants, whereas the proportion of neo-containing viruses increased 500-fold relative to wild-type virus under G418 selection. The strength and utility of p35 as a selectable marker was further demonstrated by the construction of AcMNPV expression vectors using polyhedrin-based transfer plasmids that contain p35. Recombinant viruses with foreign gene insertions at the polyhedrin locus accounted for 15 to 30% of the transfection progeny. The proportion of desired viruses was increased to greater than 90% by linearizing the parental virus DNA at the intended site of recombination prior to transfection. These results indicate that p35 and neo facilitate the selection of baculovirus recombinants and that p35, in particular, is an effective marker for the generation of AcMNPV expression vectors.     

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.     

The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells.

The p35 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is required to block virus-induced apoptosis. The trans-dominant activity of p35 suppresses premature cell death and facilitates AcMNPV replication in a cell line- and host-specific manner. To characterize the p35 gene product (P35), a specific polyclonal antiserum was raised. As revealed by immunoblot analyses of wild-type AcMNPV-infected cells, P35 appeared early (8 to 12 h) and accumulated through the late stages of infection (24 to 36 h). Biochemical fractionation of cells both early and late in infection and indirect immunochemical staining demonstrated that P35 localized predominantly to the cytosol (150,000 x g supernatant); comparatively minor quantities of P35 were associated with intracellular membranes. The cytoplasmic localization of P35 was independent of virus infection. The functional significance of the early and late synthesis of P35 was examined by constructing recombinant viruses in which the timing and level of p35 expression were altered. Delaying P35 synthesis by placing p35 under exclusive control of a strong, very late promoter failed to suppress intracellular DNA fragmentation and apoptotic blebbing in most cells. Thus, earlier expression of p35 was required to block virus-induced apoptosis. Site-specific mutagenesis of the p35 promoter demonstrated that low levels of P35 were sufficient to block apoptosis, whereas higher levels were required to maintain wild-type virus gene expression. Consistent with an early role in infection, P35 was also detected in the budded form of AcMNPV. Because of the lack of sequence similarity and its cytosolic targeting, P35 may function in a manner that is mechanistically distinct from other apoptotic regulators, including Bcl-2 and the adenovirus E1B 19-kDa protein.     

Expression of the Autographa californica nuclear polyhedrosis virus apoptotic suppressor gene p35 in nonpermissive Spodoptera littoralis cells.

Apoptosis was postulated as the main barrier to replication of the Autographa californica nuclear polyhedrosis virus (AcMNPV) in a Spodoptera littoralis SL2 cell line (N. Chejanovsky and E. Gershburg, Virology 209:519-525, 1995). Thus, we hypothesized that the viral apoptotic suppressor gene p35 is either poorly expressed or nonfunctional in AcMNPV-infected SL2 cells. These questions were addressed by first determining the steady-state levels of the p35 product, P35, in AcMNPV-infected SL2 cells. Indeed, very low levels of P35 were found in infected SL2 cells in comparison with those in SF9 cells. Overexpression of p35, in transient-transfection and recombinant-virus infection experiments, inhibited actinomycin D- and AcMNPV-induced apoptosis, as determined by reduced cell blebbing and release of oligonucleosomes and increased cell viability of SL2. However, SL2 budded-virus (BV) titers of a recombinant AcMNPV which highly expressed p35 did not improve significantly. Also, injection of S. littoralis larvae with recombinant and wild-type AcMNPV BVs showed similar 50% lethal doses. These data suggest that apoptosis is not the only impediment to AcMNPV replication in these nonpermissive S. littoralis cells, and probably in S. littoralis larvae, so p35 may not be the only host range determinant in this system.     

Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus

To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque assays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides.     

Abnormal formation of polyhedra resulting from a single mutation in the polyhedrin gene of Autographa californica multicapsid nucleopolyhedrovirus

A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.     

The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process.

Alphavirus genomes encode a small hydrophobic protein of 6 kDa (the 6K protein) that is expressed as part of a large polyprotein containing the sequences of the two virus transmembranal glycoproteins which form the spikes of the infectious particle. Although made in amounts equivalent to those of the glycoproteins, very little of the 6K protein is found in secreted infectious virions. The role of this protein in virus replication and structure has been studied by use of a variety of mutationally altered forms of 6K, which yield phenotypically distinct viruses. A complete deletion of the gene encoding the 6K protein (delta 6K) of Semliki Forest Virus (SFV) has been constructed from an SFV infectious cDNA and the transcribed RNA-produced progeny virus that closely resembled the normal virus (P. Liljeström, S. Lusa, D. Huylebroeck, and H. Garoff, J. Virol. 65:4107-4113, 1991). Further studies of this mutant have now been performed, and they show that growth of delta 6K has a strong dependency on its host cell, varying from 2 to 50% of the rate of formation of the wild-type SFV. Mammalian cells are much more defective than insect and avian cells in replication of the delta 6K mutant. This mutant is not defective in formation and transport of the glycoproteins or in production of nucleocapsids, which accumulate at the plasma cell membrane in infected BHK cells. The major defect, thus, is in the final assembly and budding of new virus. In BHK cells infected with the delta 6K strain, a relatively large fraction of the total infectious virus formed can be recovered by osmotic lysis of exhaustively washed cells. Infectious SFV totally lacking 6K is identical to wild-type SFV in the early stages of virus replication, i.e., binding and uptake. The particles themselves are more thermolabile than those of wild-type SFV, suggesting that the 6K protein may be a part of the structure of wild-type virus or that the slower budding leads to an altered configuration of the trimeric spikes. These data support other studies that implicate the 6K protein as an important but nonessential component in the assembly and budding of the alphavirus particle, perhaps by affecting the packing of the glycoproteins and their interactions with membrane lipid.     

Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection.

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.     

An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs.

Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA.     

Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant gag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.     

The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome is described. In this construct SV40 regulatory sequences, including the early promoter/enhancers and origin of DNA replication, were substituted for the AAV p5 promoter, which normally controls expression of the AAV rep gene. The hybrid genome was phenotypically indistinguishable from wild-type AAV in human cells in the presence or absence of helper virus. Upon transfection into cos-7 cells, which constitutively produced the SV40 tumor antigen, the genome replicated as a plasmid when the SV40 origin was used, although with a low efficiency compared with that of a non-AAV/SV40 replicon. The low level of replication was due to an inhibitory effect of an AAV rep gene product and was specific for replicons containing AAV sequences. Target AAV sequences required for inhibition by rep appeared to reside in the terminal repetitions since deletion of these sequences allowed efficient replication in the presence of the rep gene. The possible role for negative autoregulation of AAV DNA replication in latent infection and helper-dependent replication by AAV is discussed.