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A. Faggioni - Control of latency and replication of Epstein-Barr virus
293/∆BFLF2 cells, but was expressed in the BFLF2
recomplemented cells. On the contrary, no variations
in the expression of the other genes was observed,
confirming that BFLF2 deletion does not modify the
lytic gene cascade, which is completed with the
expression of the structural late proteins gp350/220
and BLRF2. Furthermore, to analyze if the absence
of BFLF2 might influence one of the earliest phases
of the lytic cycle, the viral DNA replication, we performed
a Gardella gel analysis which discriminates
between the two viral DNA forms, the episomal typical
of latent infection, and the linear present during
viral replication. The results showed that the recombinant
virus present in the 293/∆BFLF2 cells is able
to replicate, since newly synthesized linear genomic
DNA is produced to levels comparable to that from
other control cell lines. Finally, to analyze the infectivity
of ∆BFLF2 virus, supernatants from cells containing
the mutant virus were used to superinfect
Raji cells or to infect B lymphocytes from healthy
donors. The infection rate has been measured quantifying
GFP expression, present in the recombinant
virus genome. Approximately 400 fold less virus was
produced by ∆BFLF2 virus-infected cells, in comparison
to the control cell lines, wild type and recomplemented.
This suggests a reduced production of
virions. To analyze the intracellular maturation of
the mutant virus and to evaluate at which level this
process is blocked, electron microscopy evaluation of
the infected cells was performed. The ultrastructural
analysis showed in ∆BFLF2 cells numerous type B
capsids, devoid of viral DNA, which accumulate
within the nucleus and do not reach the cytoplasm.
In cells where BFLF2 function has been recomplemented,
the presence of numerous mature type C
virions, which fully completed the viral replication
cycle, has been consistently observed. Overall, the
above results suggest that, in absence of BFLF2, the
encapsidation of viral DNA is defective, and the
egress of virions from the nucleus to cytoplasm is
blocked, similarly to what we described previously in
cells infected with the virus deleted of the other
early lytic gene, BFRF1 (Farina et al., J Virol. 2005).
In another part of the project, we identified and
characterized p33, the product of Kaposi’s sarcoma
associated herpesvirus (KSHV) ORF69, positional
homolog of EBV BFLF2 (UL31). p33 is expressed
upon induction of viral lytic cycle with early kinetics.
Immunofluorescence analysis revealed that, in
infected cell lines, the protein is localized in the
nucleus, both in dotted spots or along the nuclear
10
membrane. Nuclear fractionation experiments
showed that p33 partitions with the nuclear matrix,
and both immunoblotting of purified virions and
immunoelectron microscopy indicated that the novel
protein is not a component of the mature virus.
Following ectopic expression in KSHV negative
cells, the protein was never associated with the
nuclear membrane, suggesting that p33 needs to
interact with additional viral proteins to reach the
nuclear rim. In fact, after cotransfection with the
ORF67 gene, the KSHV positional homolog of
UL34, the p33 intranuclear signal changed, and the
two proteins colocalized on the nuclear membrane. A
similar result was obtained when ORF69 was
cotransfected with BFRF1, the Epstein-Barr virus
(EBV) positional homolog of UL34 and ORF67.
Finally, upon cotransfection, ORF69 significantly
increased nuclear membrane reduplications induced
by BFRF1.
In conclusion, these results indicate that p33 shares
many similarities with its EBV homolog BFLF2 and
suggest that, in human γ-herpesviruses, cross-complementation
is possible between KSHV and EBV
proteins. EBV and KSHV coinfect the majority of
PEL cell lines, and molecular interactions between
the two viruses have been reported. Whether these
interactions might be relevant for the pathogenesis
of EBV and KSHV associated diseases will deserve
further studies.
Selected publications:
Serafini B, Rosicarelli B, Franciotta D, Magliozzi R,
Reynolds R, Cinque P, Andreoni L, Trivedi P, Salvetti
M, Faggioni A, Aloisi F. Dysregulated Epstein-Barr
virus infection in the multiple sclerosis brain. J Exp
Med. 2007, 204:2899-912.
Granato M, Feederle R, Farina A, Gonnella R,
Santarelli R, Hub B, Faggioni A, Delecluse HJ.
Deletion of Epstein-Barr virus BFLF2 leads to
impaired viral DNA packaging and primary egress
as well as to the production of defective viral particles.
J Virol. 2008, 82:4042-51.
Santarelli R, Farina A, Granato M, Gonnella R, Raffa
S, Leone L, Bei R, Modesti A, Frati L, Torrisi MR,
Faggioni A. Identification and characterization of the
product encoded by ORF69 of Kaposi's sarcoma-associated
herpesvirus. J Virol. 2008, 82:4562-72.