download report - Istituto Pasteur

P a r t i c i p a n t s :
Giuseppe Ragona, Antonio Angeloni, Pankaj Trivedi,
professors; Roberta Santarelli, Mara Cirone, researchers;
Antonella Farina, Roberta Gonnella, post-doc fellows;
Marisa Granato, Eleni Anastasiadou, PhD students;
Claudia Zompetta, technician.
Report of activity
In herpesviruses, viral production results from a
coordinated process requiring sequential waves of
protein synthesis (immediate-early, early and late
genes) that enable newly replicated DNA to be packaged
into the pre-assembled capsid and its internal
nucleocore. These immature viral particles are then
submitted to several rounds of envelopment-deenvelopment
that allow sequential crossings of intraand
extracellular membranes to reach the extracellular
milieu. The first egress results in a crossing of
the nuclear membrane. Previous work using mutants
of the HSV1 and pseudorabies virus has shown that
this process requires interaction between the two
conserved among herpesvirus membrane proteins
UL34 and phosphoprotein UL31. These two proteins
are sufficient to induce formation of primary
envelopes derived from the nuclear membrane in
which nucleocapsids are enwrapped after primary
egress and genetic mutants lacking one of these proteins
are sequestered in the nucleus. We suggested
recently a similar scenario also for EBV, based of the
identified interaction between the UL34 positional
homolog BFRF1 and the UL31 positional homolog
BFLF2 and on the observation that most BFRF1
mutant viruses (∆BFRF1) remain trapped in the
nucleus (Farina et al., J Virol. 2005; Gonnella et al., J
Virol. 2005). However, the weak homology at the protein
level between UL31 and BFLF2, their different
timing of expression, with BFLF2 being expressed
early and UL31 expressed late during lytic replication,
suggest functional divergence between both
Principal investigator: Alberto Faggioni
Professor of General Pathology
Dipartimento di Medicina Sperimentale
Tel: (+39) 06 4461500; Fax: (+39) 06 4454820
alberto.faggioni@uniroma1.it
9
Molecular biology of microorganisms and viruses - AREA 1
Control of latency and replication of Epstein-Barr virus
proteins. To clarify these issues, we have generated a
mutant virus devoid of the BFLF2 gene and analyzed
its phenotypic traits during lytic replication,
infection and B cell immortalization in vitro.
To construct a recombinant virus devoid of the
BFLF2 gene we used the insertion mutagenesis technology.
This is based on homologous recombinations
events which allow to disrupt or delete the gene of
interest. To this purpose, we used: 1) a BAC vector,
named p2089, where the entire genome of EBV
derived from B95-8 cell line was cloned. This vector
contains the chloramphenicol resistence gene, a bacterial
origin of replication needed for its replication
in E. coli, the GFP gene and the gene conferring
resistence to hygromycin, the selection marker for
eukaryotic cells; 2) pKD46 plasmid, which contains
the three genes gam, bet and exo used by λ phage during
homologous recombination events; 3) a linear
targeting vector containing the kanamycin
resistence gene, flanked by sequences of approximately
40 nucleotides and homologues to the regions
upstream and downstream the BFLF2 gene. In addition,
the kanamycin cassette is flanked by FRT
sequences (Flip Recombination Target) which are
recognized by the enzyme Flip recombinase during
the excision process of the cassette. In the next step,
the viral genome of the recombinant virus deleted of
the BFLF2 gene has been stably transfected in the
EBV negative cell line 293, which is known to be permissive
to EBV replication. To analyze if BFLF2
deletion alters the expression of other lytic viral
genes, the line 293/∆BFLF2, and the control cell
lines 293/WT (containing the entire viral genome
wild type) and 293/∆BFLF2+BFLF2 (recomplemented,
where BFLF2 is furnished in trans) have
been transfected with BZLF1 to induce viral replication,
and analyzed at 96 hrs post transfection by
western blot and indirect immunofluorescence to
verify the expression of early (BFRF1 and EA-D)
and late (gp350/220 and BLRF2) lytic genes. As
expected, BFLF2 was not expressed in