Examining Merkel Cell Polyomavirus Minor Capsid Proteins
An electron micrograph of MCV capsids that shows the knobby exterior of the virus
Merkel cell polyomavirus (MCV or MCPyV) is a recently discovered member of the viral family Polyomaviridae. It is a skin-dwelling polyomavirus species that appears to cause a rare but highly lethal form of skin cancer called Merkel cell carcinoma (MCC). Despite MCC being uncommon, chronic MCV infection of human skin is widespread, and most infected people have no known symptoms. The surface of polyomavirus virions is made up of pentameric knobs of the major capsid protein VP1. VP1 enables attachment of the virus to the cell surface, permitting infectious entry and delivery of the viral genome to host cells. The VP1 protein of previously studied polyomaviruses, such as simian virus 40 and murine polyomavirus, associates with two minor capsid proteins, VP2 and VP3, which are considered to play important roles during the infectious entry process.
The discovery of MCV has sparked renewed interest in polyomaviruses. There have been considerable advances in deep sequencing technologies that have led to the identification of additional human and animal polyomaviruses. In addition, the increased analyses of this viral family have revealed significant sequence differences between members. One such difference is that MCV belongs to a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. In almost all cases where either the VP2 or VP3 capsid proteins are deleted, infection or plaque formation by the virus is reduced or abolished. The mechanism by which the minor capsid proteins facilitate infection remains unclear. In addition, it is not known if MCV encodes an unusual VP3 protein or entirely lacks a VP3 protein. Christopher Buck, Ph.D., and Rachel Schowalter, Ph.D., in the Tumor Virus Molecular Biology Section of CCR’s Laboratory of Cellular Oncology, set out to determine whether the expression and function of the MCV minor capsid proteins might differ from more extensively studied members of the polyomavirus family. They studied the expression and function of the MCV VP2 and VP3 proteins of MCV using both pseudoviruses and native MCV virions grown in cell culture.
The researchers began their investigation by studying the minor capsid protein composition of MCV virions using a method by which native MCV virions can be produced from 293TT cells transfected with MCV genomic DNA. The 293TT method can also be used for intracellular production of recombinant MCV-reporter vectors, termed “pseudovirions”. Using western blotting techniques, the researchers detected a strong VP2 band but no visible VP3 bands in MCV-infected cells. MCV virions produced using the native viral regulatory sequences did not contain detectable amounts of VP3 capsid protein, making MCV unique compared to previously studied polyomaviruses. In order to dismiss the possibility that undetectably low levels of VP3 might play a role in MCV infection, possible VP3 methionine residues in native MCV virions were mutated. These mutations resulted in less than a two-fold decrease in infectivity of the viruses, demonstrating that loss of potential to express VP3 has a minimal effect on MCV infectivity. Additionally, sequence analyses suggested that more than a quarter of known polyomavirus species share MCV’s lack of VP3 protein.
The researchers then explored the role and importance of the MCV VP2 protein. Removing VP2 expression resulted in a greater than 100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. MCV pseudovirions were produced by expression of VP1 in the absence of VP2, with a low level of VP2, or with a high level of VP2. When pseudovirion preparations were inoculated onto 293TT cells, a VP2 dose-dependent effect on infectivity was observed. When a number of other cell lines that had previously been shown to be highly transducible by MCV were challenged with pseudovirus containing varying levels of VP2, the researchers made the discovery that the effect of VP2 on MCV pseudovirus transduction efficiency differed dramatically from one cell type to the next. In the cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function.
Taken together, the findings of this work indicate that the MCV VP2 gene represents a large clade of polyomaviruses that lack a VP3 minor capsid protein, which is significantly different from previously studied polyomaviruses. Knowing that this difference exists between MCV and other polyomaviruses is important for future studies that seek to understand the pathobiology of MCV and may facilitate efforts to prevent this common, and sometimes pathogenic, infection.
Summary Posted: 09/2013
Schowalter RM, Buck CB. The merkel cell polyomavirus minor capsid protein. PLoS Pathog. 2013 Aug PubMed Link