Category:Team Team TATAboxxx

Figure 1 shows that HCMV UL48 has deubiquitinase activity. A UL48 wild type was isolated as well as two point mutants that had amino acid substitutions in the presumed active site. They also generated several fusion proteins. All of these had GST at their amino ends and were tagged. One of these fusion proteins contained ubiquitin (Ub) . Figure 1C shows that when expressed UL48 was incubated with the fusion proteins, it cleaved GST-Ub-HA to produce GST-Ub, but it did not cleave any of the other proteins. This was measured using gel electrophoresis. When the two active-site UL48 mutatns were tested in figure 1D, not activity was detected, showing that their ubiquitinase activity was impaired. Protein staining in figure 1E showed that there were comparable amounts of the WT and mutant UL48 proteins in each assay, so the differences in activity can't be attributed in differences in amount of protein present.

Figure 3 shows the results demonstrating UL98's endonuclease activity. There is a wild type (WT) UL98 strain and five mutants in which alanine was used to substitute another residue within the gene. The expressed proteins were purified and labelled.

In figure 3a, the WT and mutants were incubated with closed-circular plasmid DNA to see if the gene products would convert it to open-circular and linear forms using endonuclease activity. Following incubation, gel electrophoresis measured the leftover DNA products. The WT and one of the mutants were able to degrade the entire DNA substrate by nicking the supercoiled substrate and processing the products into smaller fragments not visible on the gel. Other mutants were only able to nick the supercoiled DNA, but not digest anything else. In figure 3b, the WT and mutants were incubated with a ssDNA substrate that had a 3' quencher and a 5' fluorophore. Endonucleolytic cleavage would result in detectable fluorescence. Over time, more protein was added to the reaction in increments. The WT and one of the mutants produced significantly more fluorescence than the rest of the mutants. This indicates that those mutants suffered from impaired endonuclease function. The R164A mutant had a slightly reduced endonuclease activity compaired to the WT.

Figure 2 shows the results demonstrating UL98's exonuclease activity. There is a wild type (WT) UL98 strain and five mutants in which alanine was used to substitute another residue within the gene. The expressed proteins were purified and labelled. They were then combined with 14C-labelled DNA. The amount of cleaved DNA was measured over time. Figure 2a shows the significantly different cleavage rates between the WT gene and the R164A mutant, whose function was greatly impaired. Figure 2b show the total cleavage done by the WT and all the mutants over the course of an hour. The WT gene product exhibited significantly more exonuclease activity than the UL98-impaired mutants.

HE in human coronavirus plays a significant role in the production of an infectious virus, even though the S protein is the main viral factor in coronavirus infection. Figure 4 demonstrates the significant role HE also plays in the production of an infectious viral product. When a functional HE was added to the mutated transfectant, more viral antigens were produced. Figure 4C demonstrates the larger amount of viral antigens present in the transfectants with a functional HE compared to the pcDNA transfectant. Figure 4D demonstrates the significant amount of viral antigens who originally had a mutated HE protein were transfected with a functional HE protein. Therefore, it is apparent that the HE protein plays a significant role in producing infectious viruses.

In neurotropic strains of the herpes simplex virus (HSV), DNA is transported from the neuron body to the axon terminus. The protein Us9 is involved in the regulation of the genome transport. Figure 2 compares the percentage of transported DNA in cells containing wild type Us9 protein, and two other cells containing different Us9 gene mutations. Figure 2A demonstrates the significant effect the Us9 mutation has on DNA transport on day 3,4, and 5. Figure 2B, a different strain of HSV, demonstrates the significant effect the mutated Us9 protein has on genome transport as well, although the results were only statistically significant for day 3. None the less, the figure demonstrates that Us9 is significantly involved in the transport of the viral genome in neurotropic strains of HSV.

Figure 2A directly displays the results of BARF1-trasnfected HaCaT cell growth over 16 days in comparison to vector alone-transfected cells. BARF1 is a secreted protein that can be a powerful mitogen. In the graph shown in figure 2A, three BARF1-transfected cell cultures display more than a two-fold increase in cell division compared to vector-transfected and vector free cell cultures. Figure 2B displays the presence of the BARF1 fusion peptide of the protein in the BARF1-transfectant cells which is not present in vector-transfectant cells. This figure demonstrates the role BARF1 plays in positively regulating cell division.

Figure 1 A-C: Three different cell lines were transfected with plasmids for the over-expression of Na (which is the product of gene BRRF1) as well as control plasmids with no over-expression of Na. To see if Na initiated lysis in the cells in which Na was overproduced, immunoassays were performed to search for lytic cell products. The proteins searched for include BMRF1, Z protein, R protein, Na (through FLAG), and β-actin. In all three types of cells with plasmids over-expressing BRRF1, these lytic proteins were found in a greater amount than in the cells without the over-expression plasmids. Expression of BRRF1 leads to a product that affects maintenance of viral latency.

The evidence code is IMP because there is over-expression of the gene. Presence of the gene product resulted in an increase in specific lytic proteins measured by an immunoblot assay.

Figure 4 presents the role of BALF1 in regulating apoptosis. After being placed in an environment to cause serum starvation-induced apoptosis, the control pcDNA transfectants showed a statistically significant higher number of apoptotic control cells in comparison to BALF1 transfectants (4A). Part 4B shows 38.9% of apoptotic pcDNA-transfectants, which was about 4-10 fold increase from BALF1-transfectants. Figure 4C represents that the BALF1 expressing cells showed a much higher survival rate in serum starvation conditions which seems to be attributable to suppression of apoptosis.