Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome

An examination of Autographa californica nuclear polyhedrosis virus DNA revealed the presence of five interspersed regions, rich in EcoRI restriction sites, which shared homologous sequences. These homologous regions (hr), designated hr₁ to hr₅, occur at or near the following EcoRI fragment junctions: hr₁EcoRI-B—EcoRI-I (0.0 map units); hr₂, EcoRI-A—EcoRI-J (19.8 map units); hr₃, EcoRI-C—EcoRI-G (52.9 map units); hr₄, EcoRI-Q—EcoRI-L (69.8 map units); and hr₅, EcoRI-S—EcoRI-X (88.0 map units). Four of these regions were identified, by cross-blot hybridization of HindIII-restricted A. californica nuclear polyhedrosis virus DNA, to be within the HindIII-A/B, -F, -L, and -Q fragments. The location of these regions and the identification of a fifth homologous region were confirmed, and their characterization was facilitated, by using two plasmids with HindIII-L or -Q fragment insertions, which contained the homologous regions hr₂ and hr₅, respectively. The sizes of the homologous regions were about 800 base pairs for hr₂, 500 base pairs for hr₅, and less than 500 base pairs for hr₁, hr₃, and hr₄. A set of small EcoRI fragments (EcoRI minifragments) which ranged in size from 225 to 73 base pairs were detected in A. californica nuclear polyhedrosis virus DNA and HindIII-L and -Q fragments by polyacrylamide gel analysis. Some of the minifragments in viral DNA were present in extramolar amounts and corresponded in size to some of the minifragments present in HindIII-L and -Q. Clones of some of the EcoRI minifragments were used as probes in hybridizations to digests of viral DNA and of HindIII-L and -Q. The hybridization data, obtained under various levels of stringency, suggested that there was a degree of mismatching between the sequences which were responsible for the homology.     

Negative and positive regulation by a short segment in the 5'-flanking region of the human cytomegalovirus major immediate-early gene.

To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells. However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three- to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negative modulates expression in nonpermissive cells but positively influences expression in permissive cells.     

Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection.

The promoter-regulatory regions from the herpes simplex virus type 1 (HSV-1) gene for the immediate-early, 175,000-molecular-weight (175K) protein and the HSV-2 delayed-early gene for a 38K protein were linked to the readily assayable bacterial gene for the enzyme chloramphenicol acetyltransferase (CAT). Unexpectedly, in measurements of the constitutive expression of the recombinant genes 40 to 50 h after transfection of Vero cells, enzyme levels expressed from the delayed-early 38K-promoter-CAT construct (p38KCAT) were at least as high as those from the immediate-early 175K-promoter-CAT construct (p175KCAT). In contrast, enzyme levels expressed after transfection of a similar recombinant gene containing a second delayed-early promoter region, that of the HSV-1 thymidine kinase gene, were ca. 20-fold lower. The amounts of enzyme expressed from both p38KCAT and p175KCAT could be increased by up to 20- to 40-fold after infection of the transfected cells with HSV. In comparison, virus infection had no significant effect on enzyme levels expressed from recombinant CAT genes containing the simian virus 40 early promoter region, with or without the 72-base-pair enhancer element. Experiments with the temperature-sensitive mutants HSV-1 tsB7 and HSV-1 tsK indicate that induction of expression from p175KCAT was mediated by components of the infecting virus particle, whereas that from p38KCAT required de novo expression of virus immediate-early proteins. In addition, we show that functions required to induce expression from both p175KCAT and p38KCAT could also be provided by infection with pseudorabies virus and cytomegalovirus.     

Tightly Regulated Expression of Autographa californica Multicapsid Nucleopolyhedrovirus Immediate Early Genes Emerges from Their Interactions and Possible Collective Behaviors

To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network.     

Mouse Mammary Tumor Virus Superantigen Expression in B Cells Is Regulated by a Central Enhancer within the pol Gene

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. The mechanism of superantigen expression from the viral sag gene in B cells is largely unknown, due to problems with detection and quantification of these low-abundance proteins. We have established a sensitive superantigen-luciferase reporter assay to study the expression and regulation of the MMTV sag gene in B-cell lymphomas. The regulatory elements for retroviral gene expression are generally located in the 5′ long terminal repeat (LTR) of the provirus. However, we found that neither promoters nor enhancers in the MMTV 5′ LTR play a significant role in superantigen expression in these cells. Instead, the essential regulatory regions are located in the pol and env genes of MMTV. We report here that maximal sag expression in B-cell lines depends on an enhancer within the viral pol gene which can be localized to a minimal 183-bp region. Regulation of sag gene expression differs between B-cell lymphomas and pro-B cells, where an enhancer within the viral LTRs is involved. Thus, MMTV sag expression during B-cell development is achieved through the use of two separate enhancer elements.