The UL8 component of the herpes simplex virus helicase-primase complex stimulates primer synthesis by a subassembly of the UL5 and UL52 components.

The herpes simplex virus type 1 (HSV) UL5, UL8, and UL52 proteins form a helicase-primase complex in infected cells. Several laboratories have demonstrated that helicase and nucleoside triphosphatase activities of the heterotrimer (UL5/8/52) are indistinguishable from that of a subassembly of UL5 and UL52 (UL5/52). Although the UL5/52 subassembly functions in coupled primase-polymerase assays on homopolymeric templates, its activity on natural DNA templates has been reported to require UL8. To determine the role of UL8 in primase assays, the activity of the UL5/52 subassembly was compared to that of the heterotrimer reconstituted by adding UL8 to UL5/52. We detected significant activity of the UL5/52 subassembly in coupled primase-polymerase and oligoribonucleotide primer synthesis assays on phi X174 and M13 virion DNAs. However the addition of UL8 to UL5/52 stimulated this activity in a dose-dependent manner. We demonstrate that stimulation occurred at the level of primer synthesis. UL8 did not affect the amount or size of primers annealed to template, their utilization by DNA polymerase, or the use of specific initiation sites within the template. In kinetic studies, the rate of primer synthesis was increased by UL8 but the Km for phi X174 DNA template was unchanged. These results suggest that a function of the UL8 component of the HSV helicase-primase complex is to increase the efficiency of primer synthesis by UL5/52.

Deletions of the carboxy terminus of herpes simplex virus type 1 UL42 define a conserved amino-terminal functional domain.

The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.

Mutations in the C terminus of herpes simplex virus type 1 DNA polymerase can affect binding and stimulation by its accessory protein UL42 without affecting basal polymerase activity.

We have analyzed the effects of mutations in the herpes simplex virus type 1 DNA polymerase (Pol) C-terminal UL42 binding domain on the activity of Pol and its ability to form complexes with and be stimulated by UL42 in vitro. Wild-type Pol expressed in Saccharomyces cerevisiae was both bound and stimulated by UL42 in vitro. C-terminal truncations of 19 and 40 amino acids (aa) did not affect the ability of Pol to be stimulated by UL42 in vitro. This stimulation as well as basal Pol activity in the presence of UL42 was inhibited by polyclonal anti-UL42 antiserum, thus indicating a physical interaction between Pol and UL42. Removal of the C-terminal 59 aa of Pol and internal deletions of 72 aa within the Pol C terminus eliminated stimulation by UL42. None of the truncations or deletions within Pol affected basal polymerase activity. In contrast with their ability to be stimulated by UL42, only wild-type Pol and Pol lacking the C-terminal 19 aa bound UL42 in a coimmunoprecipitation assay. These results demonstrate that a functional UL42 binding domain of Pol is separable from sequences necessary for basal polymerase activity and that the C-terminal 40 aa of Pol appear to contain a region which modulates the stability of the Pol-UL42 interaction.

Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae.

An auxotrophic mutant of Methanococcus voltae was isolated that required coenzyme M (CoM) for growth. With the mutant as a recipient, conditions were developed that allowed the introduction of wild-type CoM+ DNA into the mutant methanogen via electroporation. This method also allowed the rescue of both a histidine and purine auxotroph as well as the introduction of DNA determining resistance to the CoM analog 2-bromoethanesulfonic acid. Electroporation of the CoM(+)-determining DNA was 50- to 80-fold more efficient than natural transformation.

Selection of a chemically defined medium for culturing fetal mouse small intestine.

We evaluated six commercially available tissue culture media in their capacity to support villi morphogenesis and enterocyte differentiation during duodenal development of the fetal mouse in vitro: McCoy's 5A, Medium 199, Swim's S77, Trowell T8, Leibovitz L-15, and RPMI-1640. The duodenal segments were resected at 15 d gestation, before the formation of intestinal villi. In the segments cultured with the first four media, no villi differentiated even at 72 h culture. The number of epithelial cells per transverse section of the explants did not increase at 24 h and thereafter the number of epithelial cells decreased, except with McCoy's 5A. With the Leibovitz and RPMI media, rudimentary villi differentiated at 24 h of culture and they attained their longest length at 48 h. With the RPMI medium, the number of epithelial cells doubled at 24 h of culture and with Leibovitz medium it doubled at 48 h. At the fine structural level absorptive cells remained poorly differentiated with all the media studied. Goblet cells were easily identified after 24 h culture; they had a well developed rough endoplasmic reticulum and numerous mucous granules. Endocrine cells differentiated in culture and they were loaded with secretion granules. It was concluded that the small intestine of the fetal mouse can be kept in organ culture for at least 72 h. Full maturation of absorptive cells seemed to require some additional factor(s) as they remained poorly differentiated with all the media studied. Because well differentiated endocrine cells were present in all the explants, it appeared that gastrointestinal hormones do not affect villi morphogenesis and absorptive cells differentiation.