Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication.     

The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1

While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early pe38 gene. However, no apoptotic activity was observed in cells transfected with pe38 alone, even when placed under the control of a constitutive promoter. Thus, pe38 was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of pe38, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection.     

The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells.

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.     

Overexpression of promyelocytic leukemia protein precludes the dispersal of ND10 structures and has no effect on accumulation of infectious herpes simplex virus 1 or its proteins

A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing the M(r) 69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of alpha, beta, or gamma groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the alpha 0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication.     

Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise co-localizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.     

Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9

The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.     

HSV-1 IE protein Vmw110 causes redistribution of PML.

Herpes simplex virus immediate-early protein Vmw110 is required for fully efficient viral gene expression and reactivation from latency. At early times of viral infection, Vmw110 localizes to discrete nuclear structures (known as ND10, PODs or Kr bodies) which contain several cellular proteins, including PML. Interestingly, the unregulated growth of promyelocytic leukaemia cells is correlated with disruption of the normal state of ND10. In this paper we show that: (i) Vmw110 affects the distribution of PML in the cell; (ii) Vmw110 proteins lacking a functional RING finger zinc-binding domain cause the production of striking abnormal cytoplasmic and nuclear structures, some of which contain PML and other ND10 antigens; (iii) a mutant form of Vmw110 which is confined to the cytoplasm appears to result in cytoplasmic PML in some cells; (iv) normal interaction with the nuclear structures requires the C-terminal portion of Vmw110; (v) the C-terminal portion of Vmw110, when linked to a heterologous protein, disrupts the normal distribution of PML. The results suggest that, in normal cells, the PML protein migrates between nucleus and cytoplasm. These observations present an unexpected link between processes involved in the control of cell growth and viral infection and latency.     

Dimerization and proper degradation of BmNPV IE2 are required for efficient virus growth in larvae of Bombyx mori (Lepidoptera: Bombycidae)

The baculovirus ie2 gene is one of the immediate early genes, and its product is known to transactivate viral promoters. However, the roles of Bombyx mori nucleopolyhedrovirus (BmNPV) ie2 in insect larvae are poorly understood. Here we investigated the functions of BmNPV IE2 in cultured cells and in insect larvae using two mutant viruses, BmIE2D and BmIE2CS. BmIE2D lacks the IE2 C-terminal coiled-coil domain that is required for IE2 dimerization. The other mutant BmIE2CS expresses an E3 ligase activity-deficient IE2 derivative, which is degraded more slowly compared with wild-type IE2. We found that ie2 mutations had little effect on BmNPV infection in cultured cells, whereas budded virus and occlusion body production was significantly reduced in the hemolymph of B. mori larvae infected with ie2 mutants. These results indicate that both dimerization and proper degradation of BmNPV IE2 are crucial steps for efficient virus growth in B. mori larvae, but not in cultured cells. Oral infection assays also revealed that the infectivity of the occluded form of ie2 mutants was normal in B. mori larvae, which is inconsistent with the results reported from ie2 mutants of Autographa californica NPV. This suggests that loss of IE2 function causes virus-specific effects in host insects.     

Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection.     

Novel baculovirus expression vectors that provide sialylation of recombinant glycoproteins in lepidopteran insect cells

This report describes novel baculovirus vectors designed to express mammalian beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes at early times after infection. Sf9 cells infected with these viral vectors, unlike cells infected with a wild-type baculovirus, produced a sialylated viral glycoprotein during the late phase of infection. Thus, the two mammalian glycosyltransferases encoded by these viral vectors are necessary and sufficient for sialylation of a foreign glycoprotein in insect cells under the conditions used in this study. While some of the new baculovirus vectors described in this study produced less, one produced wild-type levels of infectious budded virus progeny.