Using Chimeric Viruses to study Kaposi’s sarcoma- associated herpesvirus pathogenesis in mice André Filipe Coimbra e Seixas Thesis to obtain the Master of Science Degree in Microbiology Supervisors: Professor Doutor João Pedro Monteiro e Louro Machado de Simas Professor Doutor Jorge Humberto Gomes Leitão Examination Committee Chairperson: Professora Doutora Isabel Maria de Sá Correia Leite de Almeida Supervisor: Professor Doutor João Pedro Monteiro e Louro Machado de Simas Members of the committee: Doutora Sofia Pinto Guia Marques November 2016
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Using Chimeric Viruses to study Kaposi’s sarcoma-
associated herpesvirus pathogenesis in mice
André Filipe Coimbra e Seixas
Thesis to obtain the Master of Science Degree in
Microbiology
Supervisors:
Professor Doutor João Pedro Monteiro e Louro Machado de Simas
Professor Doutor Jorge Humberto Gomes Leitão
Examination Committee
Chairperson:
Professora Doutora Isabel Maria de Sá Correia Leite de Almeida
Supervisor:
Professor Doutor João Pedro Monteiro e Louro Machado de Simas
Members of the committee:
Doutora Sofia Pinto Guia Marques
November 2016
i
Acknowledgements
I would like to thank Dr. Pedro Simas for accepting me in his lab, for believing in me and for all the
good advices that he gave me during the development of my work. Thank you also to Dra. Isabel Sá-
Correia for letting me choose this master thesis theme. A special thanks to Dra. Marta Pires de Miranda
for the tremendous help that she provided me and for all the patience to teach me and to advise me.
Thank you to all the former and present members of the Herpesvirus Pathogenesis lab, especially Ana
Quendera, Francesca Martin, Sofia Cerqueira and Diana Fontinha, for the friendship and help provided.
Thank you in general to the Instituto de Medicina Molecular for providing a great environment for me to
develop my work and to Instituto Superior Técnico for accepting me in the Microbiology master.
Queria agradecer aos meus colegas de mestrado por toda a amizade e ajuda que me proporcionaram
ao longo de todo o mestrado, em especial à Inês Leonardo por ser uma amiga sempre presente.
Obrigado também a todos os meus amigos, que são a minha 2ª família com a qual eu posso sempre
contar.
Obrigado a toda a minha família por estar sempre presente e por me apoiar sempre em qualquer
momento, sem hesitação. Não poderia deixar de fazer um agradecimento especial ao meu avô, que
sem ele nada disto seria possível.
ii
Abstract
Herpesvirus are one of the most ubiquitous group of viruses in the world. Kaposi’s sarcoma-
associated herpesvirus (KSHV) is one of the eight herpesvirus that infects humans, and it is the
etiological agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric
Castleman’s disease in our species. Without a good model to study KSHV, the solution resides on a
mouse equivalent virus, murine herpesvirus-68 (MHV-68). Previous work done in our lab aimed to
generate chimeric recombinant viruses, where an essential protein in KSHV latency, latency-associated
nuclear antigen (kLANA) was cloned in a MHV-68 background, substituting the mouse herpesvirus
original LANA (mLANA). It was possible to have a chimeric virus where the influence of kLANA in vivo
was addressed, despite establishing latency in the spleen with lower levels when compared to the wild
type virus. kLANA protein sequence contains an internal acidic repeat region that is poorly
characterized. Only in vitro studies were performed so far regarding this part of the protein. The objective
of this work was to test the importance of these internal regions in vivo, by generating two chimeric
recombinant viruses’: vkLANAΔ465-929 and vkLANAΔ332-929, lacking part or the entirety of the repeat
region, respectively. In vivo it is clear that vkLANAΔ465-929 has the same latency levels as the virus
expressing full length vkLANA, whilst vkLANAΔ332-929 completely failed to established latency in the
spleen. Thus, data indicates that the aspartate and glutamate (DE) (a.a. 330-464) is crucial for the
infection and for the correct latency in the spleen.
Abstract..................................................................................................................................................... ii
Resumo ................................................................................................................................................... iii
Abbreviations ........................................................................................................................................... iv
Table of contents ..................................................................................................................................... 1
and 0,5mg/mL of proteinase K) was added to each tube and left overnight at 37°C. The next day the
proteinase K was inactivated at 95°C for 5 min in a thermocycler and the samples were analysed by
real time PCR, as previously described (Pires de Miranda et al., 2008), on a Rotor Gene 6000
thermocycler from Corbett Research using fluorescent Taqman methodology and primers and probe
sets specific for the MHV-68 M9 gene: M9-F (forward) (5’- GCCACGGTGGCCCTCTA -3’), M9-R
(reverse) (5’ – CAGGCCTCCCTCCCTTTG -3’) and M9T probe (5’- 6-FAM-CTTCTGTTGATCTTCC-
MGB-3’). PCR reactions had a final volume of 25μL containing 2,5μL of cell suspension lysate prepared
21
previously, 200μM of each primer, 300μM of probe, 1x Platinum Quantitative PCR SuperMix-UDG
(Invitrogen), 5mM of MgCl2 and 1x of Rox reference dye. The cycling program consists of a melting
temperature of 95°C for 10 min followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. In each PCR
run, a dilution series of pGBT9-M9, a plasmid that contains the M9 gene, was included as a positive
control. PCR results were analysed with Rotor-Gene™ 6000 real-time rotary analyser Software version
1.7. (Corbett Life Science). For all dilutions tested each replicate eas scored positive or negative based
on comparison with negative (water sample) and positive (plasmid containing M9 gene) controls. The
frequency of cells with viral DNA was calculated according to the single-hit Poisson model (SHPM) by
maximum likelihood (Bonnefoix et al., 2001). This model assumes that only one cell is necessary and
sufficient for generating a positive response. The method consists in modelling the data from the limiting
dilution assay according to the linear log-log regression model fitting the SHPM.
3.17 Statistical analysis
Data comparisons between groups was performed by an ordinary one-way ANOVA and Mann-
Whitney test, when applied. Statistics were calculated with GraphPad Prism Software. For limiting
dilution analysis 95% confidence intervals were determined as described (Marques, S. et al., 2003).
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4. Results
4.1 Generation and characterization of MHV-68 recombinant viruses
Previous work from our laboratory shows that the chimeric kLANA MHV-68 virus (v-kLANA),
where the endogenous ORF73 (mLANA) was replaced by KSHV ORF73 (kLANA), is a viable model to
study KSHV pathogenesis in mice (Pires de Miranda and Simas, unpublished results). This model allows
to address the relevance of the different kLANA domains in an in vivo infection, by generating viral
recombinants with deletions in these domains. The internal acidic repeat region is the least
characterized domain of kLANA. Previous in vitro studies with two kLANA internal repeat deletion
mutants (kLANAΔ465-929 and kLANAΔ332-929) are expressed, in a KSHV background, have shown
that both these mutant proteins have a defect in viral episome maintenance (De León Vazquez and
Kaye, 2013).To test the importance of this internal repeat region in our in vivo chimeric model, the same
two recombinant proteins were introduced in a MHV-68 background: kLANAΔ465-929 harbours a
deletion encompassing aminoacids 465 to 929, which removes part of the glutamine region (Q), the full
leucine zipper (LZ) and the glutamate and glutamine region (EQE); kLANAΔ332-929 has a deletion in
aminoacids 332 to 929, disrupting the totality of the internal repeat region of the protein (Figure 7).
The construction of this viruses was made via a mutagenesis procedure in E. coli DH10B
according to the Two-Step-Replacement Strategy (O’ Connor et al., 1989) using a MHV-68 BAC plasmid
with the entire MHV-68 genome cloned (Collins et al., 2009). This technique allows the maintenance of
viral genome as a plasmid in E. coli, where a homologous recombination process occurs with a
subsequent reconstitution of the viral progeny by transfection of the BAC plasmid into eukaryotic cells
(Adler et al., 2003). The full procedure is described in Materials and Methods. To identify the BAC’s
harbouring the mutant kLANA sequences, a PCR with primers specific for the C-terminal region of
Figure 7: Diagram of KSHV LANA and LANA deletion mutant proteins. The numbers indicate aminoacid residues and the different domains of the protein are also indicated, namely the proline-rich (P), the aspartate and glutamate (DE), the glutamine (Q), the glutamate and glutamine (EQE), the leucine zipper (LZ) region and the DNA binding domain (DBD). The fold deficiency in episome maintenance in vitro is also shown and it was determined by comparison with the kLANA WT (De Leon Vazquez et al., 2013).
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kLANA was performed (see Material and Methods - Generation and characterization of recombinant
viruses). Furthermore, the integrity of the viral genome was analysed by restriction digestion using two
different restriction enzymes, EcoRI and BamHI (Figure 8). No unexpected changes in the restriction
profile were detected. The blue boxes show the bands specific of the v-kLANAΔ465-929, the red boxes
show the bands specific to the v-kLANAΔ332-929. Two independent clones were engineered for each
mutant (i).
The mutant BAC’s were then transfected into BHK-21 fibroblasts to reconstitute the viruses in
cells. To remove completely the BAC cassette, the reconstituted viruses were sub-cultivated with NIH-
3T3-Cre cells that through the Cre-lox system, removed the BAC sequence that is flanked by lox-P sites.
4.2 The chimeric recombinant viruses display normal in vitro growth
To assess if the mutant viruses had a normal growth kinetics, a multistep growth curve was
performed. Permissive BHK-21 fibroblasts were infected at low MOI (0.01pfu/cell) and during five
Figure 8: Assessing the integrity of the mutant BAC plasmids by restriction enzyme profile analysis using EcoRI, BamHI. The blue boxes show the bands specific of the v-kLANAΔ465-929, the red boxes show the bands specific to the v-kLANAΔ332-929. Each mutant has two independent clones.
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days cells were scrapped and the titrations were performed by plaque assay. Two different viruses
were used as controls: the WT MHV-68 that harbours the mLANA (v-WT) and the chimeric MHV-68
where mLANA was replaced by the full length kLANA (v-kLANA). It is known that the ORF73 from
the MHV-68 is not essential for in vitro growth (Fowler et al., 2003). There was no significant
difference between infection groups (one-way ANOVA) and the v-kLANA virus growth was similar
to the growth previously observed in the lab (Pires de Miranda and Simas, unpublished) (Figure 9).
This indicates that neither the substitution of the mLANA for kLANA nor the deletions in the kLANA
gene affect the lytic replication.
Figure 9: Multistep growth curve by infection of BHK-21 cells at 0.01 pfu per cell. Samples were harvested, freeze-
thawed and titers were determined by plaque assay on monolayers of BHK-21 cells.
4.3 Expression of kLANA mutant proteins
In order to investigate if all the mutants expressed the different kLANA proteins correctly, an
immunoblot was performed using antibodies against kLANA and other cellular and viral proteins.
BHK-21 cells were infected with a MOI of 3pfu/cell during 6h. LN53 is a commercial antibody that
recognizes the EQE repeats within the internal repeat region of kLANA. This is a very sensitive
antibody that recognizes multiple epitopes in the full length kLANA that are absent in the deletion
mutants. Because both mutants used lack that specific part of the protein, another commercial
antibody (4C11), that recognizes a different epitope (a.a.122-329), was ordered but it was not
sensitive enough to detect kLANA proteins under these conditions (Figure 10). kLANAΔ465-929
should have been detected with a molecular weight of approximately 140kDa and the kLANAΔ332-
929 with one of approximately 100kDa. Even the chimeric virus with the full length kLANA add a
poor recognition when compared with α-kLANA LN53. The infection levels were similar between
samples as demonstrated by the level of M3 protein and the loading control for cell infection (α-
actin) was also similar in all samples. mLANA was also detected only in the MHV-68 WT sample,
T im e p o s t- in fe c t io n (h )
Lo
g p
fu/m
L
0 2 4 4 8 7 2 9 6 1 2 0
0
2
4
6
8
v -k L A N A 3 3 2 -9 2 9 1 3
v -k L A N A 3 3 2 -9 2 9 i
v -k L A N A 4 6 5 -9 2 9 1 0
v -k L A N A 4 6 5 -9 2 9 i
v -k L A N A
v -W T
25
as expected. Clearly the problem resides in the lack of a good antibody. Serum from a patient with
Kaposi’s sarcoma, that contains polyclonal antibodies (gift from Professor Kenneth Kaye) was also
used to try to detect kLANA mutants but was also not effective in the detection (data not showed).
Figure 10: Detection of the expression of WT and mutant kLANA proteins. BHK-21 cells were infected with a MOI
of 3pfu/cell during 6h. The proteins were detected with the antibodies indicated on the right. Molecular weight (in
kDa) is indicated on the left.
4.4 Transcription analysis of kLANA mutants
Since it was impossible to detect the kLANA mutant proteins by immunoblotting, the levels of mRNA
expression were quantified instead. Because kLANA proteins affect their own transcription, these
analyses could also reveal if the lack kLANA internal regions would affect expression. BHK-21 cells
were infected with virus at a MOI of 5 pfu/cell during 8h. Total intracellular RNA was extracted, reverse
transcribed and real-time qPCR was performed using primers specific for kLANA and ORF50 viral
transcripts (see Materials and Methods - Transcription analysis of kLANA mutants). Relative kLANA
mRNA values were normalized to ORF50, a lytic gene that is transcribed during the first 8h after
infection. Ratios were calculated using the Pfaff method (Pfaff, 2001), and plotted as fold difference of
kLANA mRNA levels relative to the chimeric virus v-kLANA (Figure 11).
Analysing the data it is clear that both v-kLANAΔ465-929 independent mutants express kLANA at
the same level as v-kLANA, while v-kLANAΔ332-929 independent mutants present a deficit in kLANA
expression levels. One possible explanation for this deficit is that the aspartate and glutamate internal
26
acidic and part of the glutamine region may interact with cellular proteins with functional activity in
transcription as already discussed by others (De Leon & Kaye, 2011).
Figure 11: Quantification of kLANA transcripts. Total RNA extracted from BHK-21 infected with the corresponded
virus at an MOI of 5 pfu/cell until 8 h.p.i. Poly-A mRNA was reverse transcribed and cDNA was obtained. The
controls with no reverse transcriptase (-RT) and with mock cells were performed and both are not detectable, as
expected (results not shown). Relative mRNA values, normalized to ORF50 gene, were calculated by the Pfaffl
method taking into account the efficiency of the PCR reaction. Data are presented as fold difference in gene
expression compared to v-kLANA. Each sample was run in triplicate, and the error bars represent the standard
deviation.
4.5 The internal acidic repeat region has impact in the establishment of latency in the
spleen
It is known that the mLANA is essential for latency in the spleen (Fowler et al., 2003) and
homology between mLANA and kLANA exists (Russo et al., 1996; Virgin et al., 1997; Grundhoff
and Ganem., 2003). Furthermore, the chimeric v-kLANA is able to establish splenic latency following
intranasal inoculation (Pires de Miranda and Simas, unpublished results). It is thus essential to
discover if the kLANA mutants lacking parts of the acidic internal region can establish latency in the
spleen. An infectious centre assay was performed to quantify the levels of latent viruses in the
spleen of mice infected with the recombinant viruses (Figure 12). C57BL/6 mice were inoculated
intranasally with 104 pfu of virus and, at 14 days post-infection the mice were sacrificed, spleens
were surgically removed and processed as described in Materials and Methods – Infection of mice.
The latent load in the spleen was examined by quantification of ex vivo reactivation-competent
viruses when cultured with permissive fibroblasts. The presence of latent viruses in the splenocytes
results in the formation of viral plaques within the cell monolayer that can then be quantified (Figure
12, closed circles). To make sure that no lytic infectious viruses were present in the splenocyte
population, aliquots of splenocytes were freeze-thawed first and then co-cultured with the
kL
AN
A m
RN
A l
ev
els
re
lati
ve
to
co
ntr
ol
(fo
ld d
iffe
re
nc
e)
Co
ntr
ol (v
-kL
AN
A)
v-k
LA
NA465-9
29
v-k
LA
NA465-9
29 i
v-k
LA
NA332-9
29
v-k
LA
NA332-9
29 i
0 .0
0 .5
1 .0
1 .5
27
permissive cells. No pre-formed infectious viruses could be detected at any time point for any of the
analysed viruses (Figure 12, open circles).
As previously observed, v-kLANA established latency in the spleen albeit at lower levels when
compared with v-WT (Pires de Miranda and Simas unpublished).
In the v-kLANAΔ465-929 mutants, latency is not affected when compared to the v-kLANA.
Regarding the v-kLANAΔ332-929 mutant, however, the ability to establish latency in the spleen is
abrogated (bellow the limit of detection of this assay). This indicates that the aspartate and
glutamate internal acidic (a.a. 330-463) has influence in the ability to establish latency in the spleen.
This phenotype is unlikely to reflect inadvertent mutations introduced during the mutagenesis
process since equivalent phenotypes were observed in both independently generated mutant
viruses.
Figure 12: Quantification of latent infection in spleen 14 d.p.i. by explant co-culture plaque assay (closed circles).
Titres of infectious virus were determined in freeze/thawed splenocyte suspensions (open circles). Each circle
represents the titre of an individual mouse. Data were combined from two independent experiments each with five
mice per infection group. The dashed line represents the limit of detection of the assay. Horizontal bars indicate
arithmetic means. The comparison between v-kLANA and both v-kLANAΔ465-929 independent mutants was not
significant. ***(p<0.001) and **(p<0.01).
4.6 Quantification of the frequency of viral DNA-positive total splenocytes and GC B-
cells
To confirm the phenotype observed in the infectious centre assay, a limiting dilution assay for
quantification of DNA positive cells was performed with the total splenocyte population and with
sorted purified GC B-cells (Figures 13 and Table 1). Quantification of viral infection in GC B-cells is
highly relevant since mLANA is selectively expressed in proliferating GC B-cells (Marques et al.,
2003) and mLANA was replaced by kLANA. The viral DNA was detected by real-time PCR with
primers and probe sets specific for the MHV-68 M9 gene (see Materials and Methods - Limiting
dilution assay and Real-time PCR of viral DNA positive cells). Analysing the graphics it is clear that
1 4 d .p .i.
pfu
/sp
lee
n
v-W
T
v-k
LA
NA
vkL
AN
A465-9
29
vkL
AN
A465-9
29 i
vkL
AN
A332-9
29
vkL
AN
A332-9
29 i
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
1 4 d .p .i.
pfu
/sp
lee
n
v-W
T
v-k
LA
NA
vkL
AN
A465-9
29
vkL
AN
A465-9
29 i
vkL
AN
A332-9
29
vkL
AN
A332-9
29 i
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
***
**
**
28
the phenotype observed in the infectious centre assay is maintained. The v-WT virus has the
expected frequencies of viral DNA+ cells both in the total splenocyte population and in the GC B-
cells (Correia et al., 2013) as well as the v-kLANA despite being approximately 1log lower than the
vWT as previously observed in the lab (Pires de Miranda and Simas, unpublished).
There are no differences between v-kLANAΔ465-929 and v-kLANA and the v-kLANAΔ332-929
mutant has a frequency of viral DNA+ cells that it is not detectable both in total splenocytes (Figure
13A) and in the GC B-cells (Figure 13B). This data shows that the aspartate and glutamate internal
acidic (DE) and part of the glutamine (Q) region is crucial for the infection of GC B-cells and for
establishment of latency in the spleens in this chimeric model.
Figure 13: (A) Reciprocal frequencies of viral DNA-positive cells in total splenocytes or GC B-cells (CD19+CD95hiGL7hi) (B) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. N.D. Not detectable. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals.
a
Data were obtained from pools of 5 spleens. b
Frequencies were calculated by limiting dilution analysis with 95% confidence intervals (numbers in parentheses). c
The purity of sorted cells was determined by fluorescence-activated cell sorting (FACS) analysis and it was always greater than 95%.
A B
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5. Discussion
In this work, two kLANA-MHV68 chimeric mutant viruses were generated, v-kLANAΔ465-929,
lacking part of the Q, the LZ and the EQE internal repeat region and v-kLANAΔ332-929, lacking the
entire internal repeat region. They were created to assess the impact of kLANA internal repeat deletions
in the chimeric in vivo model. Results show that the DE internal repeat region and part of the Q region
(a.a. 330-463) are essential for the viral latency in the spleen.
Recombinant viruses displayed an in vitro growth similar to the v-WT and v-kLANA viruses
(Figure 3). This was expected since it has been reported that mLANA was not essential for in vitro
growth (Fowler et al., 2003). This indicates that neither the substitution of the mLANA for kLANA, nor
the internal repeat deletions in the kLANA gene dramatically affected the lytic replication.
In vivo analysis by infectious centre assay show that the v-kLANAΔ465-929 has a latency peak
very similar to vkLANA, in both independent clones and the v-kLANAΔ332-929 mutant has a completely
different result, with a phenotype emerging (Figure 12). For the latter, the ability to establish latency in
the spleen is below the limit of detection of the assay. This result indicates that the DE internal repeat
region, encompassed in a.a. 330-463, influences the ability of v-kLANA to establish latency in the
spleen. In agreement is the detection of the frequency of viral DNA positive cells, where no differences
between v-kLANAΔ465-929 and v-kLANA are observed, and the v-kLANAΔ332-929 mutant present a
frequency of viral DNA positive cells that it is not detectable both in total splenocytes and in the GC B
cells (Figures 13A and 13B and Table 1). The correlation between this two independent assays helps
to confirm the phenotype observed in the v-kLANAΔ332-929 mutant, where the lack of establishment of
latency in the spleen assessed by infectious centre assay is supported by the non detection of viral DNA
positive cells in total splenocytes and in GC B cells.
The results obtained in this work are in line with studies made by others, where in vitro, either
in transient transfected cells or in stable cell lines expressing similar LANA deletion mutants, kLANA
without the complete internal repeat region had deficits in maintaining viral episomes during latency
(Alkharsah and Schulz in 2011; De León Vasquez and Kaye, 2011; De León Vasquez and Kaye, 2013).
Some proteins, important for the correct replication and/or segregation of the episomes, might
interact with this internal region and the lack of that region impairs the persistence of viral genomes in
the spleen. Protein conformation itself might be altered by the lack of this structural parts, making it
impossible to function correctly. There also might be additional functions other than DNA segregation
and replication exerted by the internal repeat region that are essential for persistency of episome in the
spleen.
It was also important to detect the proteins by western blot to check if the lack of latency of the
v-kLANAΔ332-929 was due to low protein levels or lower stability of the protein. Given the fact that v-
kLANAΔ465-929 establishing latency in the spleen, with levels similar to v-kLANA, it is likely that the
protein is being expressed. The most common used antibody to detect kLANA is LN53, which is an
antibody that detects several epitopes in the EQE repeat region and is very sensitive. Because both v-
30
kLANAΔ465-929 and v-kLANAΔ332-929 lack that specific part, another antibody was used and it was
not successful in the detection (Figure 11). Serum from a KS patient was also used to try to detect the
proteins but is was unsuccessful as well. In the future efforts should be done to detect these mutant
proteins, like the construction of viruses with higher affinity epitope tags (HA tag for example), to be able
to detect these proteins with common commercial antibodies. The use of more cells or higher MOI to
intensify the detection and the analysis at different time-points after infection can also be done to
improve the detection levels in a western blot
The levels of kLANA poly-A transcripts in both mutant viruses were also assessed in infected
BHK-21 cells and compared to v-kLANA. The v-kLANA and the v-kLANAΔ465-929 presented very
similar levels of transcripts whereas the v-kLANAΔ332-929 have a slight deficit in the kLANA expression
levels (Figure 11). This might be due to the fact that the aspartate and glutamate internal acidic and part
of the glutamine region may interact with cellular proteins with functional activity in transcription (De
Leon & Kaye, 2011). Again, with the lack of such structural parts, the protein conformation might be
altered in such a way that blocks the access of the transcription machinery, making it difficult for the
correct transcription to occur. Overall these results confirm expression of the mutant kLANA transcripts
in infected cells and suggest that deletion of the internal regions does not affect significantly its own
expression.
Many proteins interact with different parts of LANA. Some were identified and mapped to overlap
with the internal repeat region. These proteins fall in the transcription category, in chromosome structure
or modification and in cell growth regulation (De León Vasquez and Kaye, 2011). Protein-protein
interaction assays should be performed to identify even more interactions and, more importantly, map
the location of those interactions. It is possible that most interactions occur within the internal repeat
region since it is the biggest region (Figure 5). By doing this kind of assays it might be possible to unveil
more functions of this internal structural part.
31
6. Conclusion
This work took advantage of the creation of an in vivo model to study the pathogenesis
associated with Kaposi’s sarcoma-associated herpesvirus infection. Two chimeric recombinant viruses
in a MHV-68 background were created, where the MHV-68 ORF73 (mLANA) was substituted by KSHV
ORF73 (kLANA): v-ΔkLANA465-929, which retains the DE region and part of the Q region and v-
ΔkLANA332-929, lacking the entire internal repeat region. The fact that these parts were missing helped
to unveil their importance in this in vivo model.
v-ΔkLANA465-929 had the same ability to establish latency in the spleen as the v-kLANA, while
the v-ΔkLANA332-929 has a big deficit in establish latency. This leads to conclude that the aspartate
and glutamate and part of the glutamine internal regions (a.a. 330-464) are somehow crucial for the
latency in the spleen. These results have similarities with in vitro results obtained by other authors where
it was seen that the lack of some internal parts of kLANA led to a deficit in the maintenance of the viral
episomes during latency.
In the future, a deletion mutant encompassing precisely aminoacids 330-463 should be
constructed, in order to prove that this is really a crucial part of the protein for infection and establishment
of latency. Furthermore, with this mutant virus, the EQE repeats are maintained, meaning that the
epitopes for the LN53 antibody are maintained and it is thus possible to detect the protein.
Future work on identification of more proteins that interact with this internal part is also needed to
elucidate better the mechanisms underlying LANA full function.
By having more knowledge about this protein, that is crucial for latency, might shed light into a possible
therapeutic target for fighting the KSHV infection.
32
7. References
. Adler, H. et al., 2000. Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an