Proto-oncogene derived from polyomavirus SV40
SV40 large T antigen (polyomavirus
SV40. TAg is capable of inducing
malignant transformation of a variety of cell types. The transforming activity of TAg is due in large part to its perturbation of the
retinoblastoma (
pRb)
[1] and
p53 tumor suppressor proteins.
[2] In addition, TAg binds to several other cellular factors, including the transcriptional co-activators
p300 and CBP, which may contribute to its transformation function.
[3] Similar proteins from related viruses are known as
large tumor antigen
TAg is a product of an early gene transcribed during viral infection by SV40, and is involved in
Papovavirus) family,
Orthopolyomavirus genus. Polyomaviruses infect a wide variety of
vertebrates and cause solid tumours at multiple sites. SV40 was isolated by Sweet and
Maurice Hilleman in 1960 in primary monkey kidney cell cultures being used to grow
Sabin OPV.
[4]Domains
The TAg has a
TP53-binding domain, a Zinc finger, and a Superfamily 3 ATPase/Helicase domain. It has two motifs, one for nuclear localization signal, the other being the LXCXE motif.
[5]Mechanism
After entering the cell, the viral genes are transcribed by host cell
cis-acting
regulatory element surrounding the origin of replication directs transcription, and T-antigen directs transcription and replication.
SV40 DNA replication is initiated by binding of large T-antigen to the origin region of the genome. The function of T-antigen is controlled by phosphorylation, which attenuates the binding to the SV40 origin. Protein-protein interactions between T-antigen and DNA polymerase-alpha directly stimulate replication of the virus genome.
T-antigen also binds and inactivates
tumor suppressor proteins (p53, p105-Rb). This causes the cells to leave G1 phase and enter into S phase, which promotes
DNA replication.
The SV40 genome is very small and does not encode all the information necessary for DNA replication. Therefore, it is essential for the host cell to enter S phase, when cell DNA and the viral genome are replicated together.
Therefore, in addition to increasing transcription, another function of T-antigen is to alter the cellular environment to permit virus genome replication.
Nuclear localization signal
The SV40 large T-antigen has been used as a model protein to study
nuclear localization signals (NLSs).
[6] It is imported into the nucleus by its interaction with
importin α.
[7] The NLS sequence is PKKKRKV.
[6]Interaction with pRb via the LXCXE motif
SV40 large TAg, other
human papillomavirus E7 proteins share a structural motif that encodes a high-affinity
pRb-binding domain.
[8][9] A diagnostic pattern for a high-affinity pRb-binding domain was refined using an
artificial intelligence pattern-induction program running on a massively parallel
supercomputer (
Connection Machine-2).
[9] The motif is characterized by an
Asp,
Asn or
Thr residue followed by three invariant amino acids, interspersed with non-conserved amino acids (designated by x, where x cannot be a
Lys or
Arg residue).
[9] A negatively charged region frequently follows carboxy-terminal to the pRb-binding domain.
[9]- {Asp/Asn/Thr} – Leu – x – Cys – x – Glu – x – ... {negatively charged region}
electrostatic properties are highly conserved in this motif. For example, a local hydrophobicity maximum occurs in the vicinity of the invariant
Leu residue.
[9] A net negative charge occurs within 3 residues amino-terminal to the invariant
Leu residue; furthermore, positively charged amino acids (
Lys or
Arg) are not found within the
Leu – x –
Cys – x –
Glu sequence, nor in the positions immediately flanking this sequence.
[9] The pRb-binding motif and negatively charged region match to a segment of SV40 TAg beginning at residue 102 and ending at residue 115 as shown below:
- – Asn – Leu – Phe – Cys – Ser – Glu – Glu – Met – Pro – Ser – Ser – Asp – Asp – Glu –
Functional studies of TAg proteins bearing
hydrogen bonds also stabilize the TAg–pRb complex.
[11] For example, the side chain of Glu-107 forms hydrogen bonds by accepting hydrogens from the main chain
amide groups of
Phe-721 and
Lys-722 in pRb.
[11] The mutation of
Glu-107 to
Lys-107 is expected to result in loss of these hydrogen bonds.
[11] Furthermore, the side chain of
Lys-107 would likely have energetically unfavorable interactions with the amide of
Phe-721 or
Lys-722,
[11] destabilizing the complex.
Strong experimental evidence confirms that positively charged amino acids (Lys or Arg) significantly weaken the binding interaction with pRB when positioned in the vicinity of the Leu – x – Cys – x – Glu sequence.[12] This is likely due to the fact that the binding surface on pRb features six lysine residues, which will tend to repel positive residues within or flanking the Leu – x – Cys – x – Glu sequence.[12]
Of note, the highest-risk oncogenic
human papillomavirus (HPV) strains (16, 18, 31, 45) encode E7 proteins featuring high-affinity pRb-binding domains which match the diagnostic pattern given above.
[9]References
