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Integrin a3b1 (CD 49c/29) Is a Cellular Receptor for Kaposi's Sarcoma-Associated Herpesvirus (KSHV/HHV-8) Entry into the Target Cells
Shaw M. Akula, Naranatt P. Pramod, Fu-Zhang Wang, and Bala Chandran
Department of Microbiology
Molecular Genetics and Immunology
The University of Kansas Medical Center
The paper describes the role of a3b1 integrin as the receptor for entry of HHV-8 into cells. The RGD motif on one of the glycoproteins of HHV-8 is highly conserved in different HHV-8 strains, thus implying its general importance. Integrin molecules, like a3b1, are heterodimeric receptors containing noncovalently-associated transmembrane a and b glycoprotein subunits (Giancotti and Ruoslahti) (Akula, 2002). The experiments suggest a role for a3b1 in the binding and entry for HHV-8 and possibly a role in Kaposis Sarcoma through FAK-mediated pathways.
The investigators did indeed test their hypothesis thoroughly. As a preliminary step they labeled HHV-8 with GFP and monitored virus entry and protein expression (ORF73 protein) in HFF cells. This is simply to ensure that the virus does indeed bind and infect HFF cells (Fig. 1). The next step was to determine the role of RGD peptides in virus binding. Figure 2A depicts a significant inhibition of virus entry when cells were incubated for one hour prior to infection with peptide sequences containing RGD. Presumably this is because the RGD peptides compete with HHV-8 for the binding sites on the HFF cells. They mention that RGDgB-N1 did not inhibit as well as the smaller proteins due to their possible aggregation, whereas the smaller ones could move around more and not clump. This seems like a reasonable conclusion. The data (not shown) which would give the reader a picture of how the RGD peptides similarly decreased GFP expression and protein expression would have been helpful. Figure 2B shows that antibodies against RGD-peptides (such as the one in HHV-8) significantly inhibited GFP-HHV-8 infection in a dose-dependent manner. The increase in antibodies and subsequent decrease in infection lends support that RGD is very important. As a control antibodies against peptides without RGD had no effect on virus binding. The only problem with this is that the anti-gB antibodies did as well as the anti-RGDgB antibodies which does not lend much to their argument. Figure 2C also has some apparent contradictions. Fibronectin (which interacts with integrins, dependent or independent of RGD), when incubated with the virus and cells, did inhibit virus infectivity at high concentrations. However, laminin, like fibronectin in respect to biding integrins, actually increased infectivity. Since it can bind to RGD, it seems as if it would act like fibronectin and decrease infectivity in the cells. The researchers simply say it is being investigated further.
Figure 3A provides them with some better results. Monoclonal antibodies were produced against eleven integrin components in cells. Antibodies bound to a3 and b1 inhibited HHV-8 by about 48% and 35%. a2b1 was not significantly different from b1, but its inhibitory qualities were credited to b1 and not a2. That is somewhat strange since they didnt even test a2 alone and could not readily say that a2 wasnt the protein having the effect. Figure 3B supports the fact that HHV-8 is inhibited by a3, b1, and a2b1. Antibodies against these integrins were made and the results show about 40 to 60% inhibition in a dose dependent manner. The control was another antibody of no relation. Since aaand b are bound in cells, Figure 3C shows the integrin a3b1 inhibiting infection about 75% at a concentration of 5 microliters per milliliter, while a5b1 had little effect. This suggests that the configuration of a3b1 itself is what the HHV-8 glycoprotein using for entry.
The researchers then determined if a3b1 is indeed present in infected HHV-8 cells. Figure 4E shows that 87% of cells had b1 and 90% a3. Figure 5A is FACS data on the level of a3 in cells with a control plasmid versus a pCDNA3-human a3 integrin cDNA. With the plasmids, the data show a huge increase in a3 integrin in the cells. Then Figure 5B shows CHO-B2 cells which have been transfected with human a3 plasmid (from the pCDNA3-human a3 integrin cDNA). As there was an increase in human a3/mouseb1 integrin, infectivity also increased. Thus human a3 and mouse b1 must be able to form the same conformation as a wild-type integrin. The increased infectivity is indeed appropriate if a3b1 in cells is the receptor for HHV-8 entry. Figure 5C uses monoclonal antibodies to a3 and b4. b4 is used as one of the controls because from the current results it should have nothing to do with HHV-8 infectivity. The other control is the CHO-B2 cells with the a3 plasmid. In the absence of the anti-a3 antibody there is more infectivity than when the antibody is introduced. This lends evidence that when a3 is open to virus binding (and not bound by an antibody) more cells will be infected. B4 had no effect.
Figure 6A investigates whether heparinaseI/III will compete for binding to HHV-8 with a3b1. Fig. 6A is a Western blot using mouse anti-gB IgG. When the virus was not present a1, a3, and b1 did not react in the immunoprecipitation. This upholds the idea that virus-integrin binding is very specific. When in the presence of the virus, a3 and 1 both immunoprecipitated, however a1 did not, as expected. Even in the presence of heparinase, the a3 and b1 bound which shows the ultimate attraction HHV-8 has with the a3b1 integrin. Figure 6B sets out to determine if a3b1 is within an attachment or post-attachment step of infection. HHV-8 binds to haparin before entry. Figure 6B shows about 90% of the HHV-8 binding to haparin over RGD or a3 or b1. This led the researchers to conclude that a3b1 has its job in entry to the cell and not binding.
It was observed that FAK (a protein-tyrosine kinase which autophosphorylates when in contact with integrin-ligand interaction) is activated on the onset of HHV-8 entry. Thus the researches wondered if the interaction between HHV-8 and the integrin a3b1 had anything to do with its activation. Figure 7 shows colocalization of FAK after HHV-8 infection. The results do indicate that FAK is altered after infection. Figure 7B (SDS-PAGE) shows the increase in levels of FAK phosphorylation after infection. Thirty minutes after infection is when FAK appears the darkest, but even five minutes after infection FAK phosphorylation as been induced. Actin bands below stand as a control. Next they want to determine if a3b1 has any bearing on the activation of FAK. Figure 7C, also SDS-PAGE, shows a dark FAK band at thirty minutes postinfection in lane 1. The HHV-8 in lanes 2-4 were incubated with 5, 2.5, and 1.25 microliters/milliliter of soluble a3b1 and then allowed to infect the cells. The lightest band appears in lane 2. This is because HHV-8 was incubated with a3b1 before infection and it has been shown that soluble a3b1 decreases infectivity. Thus FAK would not be as active in cells that were not being infected. The a5b1, as a control, appeared to have no effect on the FAK (especially if you compare actin bands, the one for a5b1 is lighter anyway.) Figure 7D is the same as 7C, however, here the researchers use anti-gB antibodies and again, FAK phosphorylation is diminished.
The research met kinks in many places. There were problems arising from the increased infectivity when in the presence of laminin and the lack of control on mutations which may have occurred in the virus strain. However, they load you with so many positive results and good data, that one cannot deny that a3b1 does indeed have a relationship with HHV-8 infection. However, the paper leaned more towards proving the HHV-8 glycoprotein and a3b1 integrin RGD-dependent relationship than with Kaposis Sarcoma. I believe that a3b1 has an intense receptor-integrin relationship with HHV-8. I also believe that a FAK pathway is jump-started by HHV-8 infection. The relationship between a3b1 and FAK is not well defined. Perhaps there is a signaling pathway that starts with a3b1 and subsequently activates FAK. As of now, I cannot defend any argument which says a3b1 is essential in FAK activation.
Future research for this is limitless. First and foremost, I see the medical benefits with making a drug using a3b1. The next step, however, is to present substantial evidence that a3b1 is the igniter of FAK phosphorylation. Perhaps one could immunoprecipitate a3b1 before infection and see if the FAK protein is bound to it in any way via itself or any other proteins. If FAK is bound to a3b1, then its intimate initiation by a3b1 is not so doubtful. HHV-8 binding to a3b1 may change its form, thus activating FAK. However, FAK may be activated by another integrin protein which aids in the entry of HHV-8. Identifying other possible entry proteins may lead to information on FAK activation. Use another envelope protein from HHV-8 and do the same thing as reported above. Thus by immunoprecipitation of another integrin, one can see if indeed FAK is attached. Another idea is to pulse-chase the FAK protein before and after infection. It was shown that FAK is activated in the first five minutes, so if you could see where FAK is when the cell is infected, you might be able to narrow down the possibilities. Perhaps FAK is in the cytoplasm and reacts to the viral DNA or perhaps it is close to the plasma membrane and is interacting with the integrin a3b1.
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