Poster01 Prof AMS 2015

Vif- H3.3 interaction, a new Vif function?

Alexandra Maia e Silva

,

Inês Cavaleiro

,

Ana Clara Ribeiro

e

Isabel Barahona

1

_CiiEM,

_{Escola Superior de Saúde Egas Moniz,}

_{Instituto Superior de Ciências da Saúde Egas Moniz}

Quinta da Granja, Monte da Caparica, 2819-511 Caparica

INTRODUCTION

HIV virus encodes for several accessory proteins in particular Vif protein that is essential in an early phase of viral infection.

In 2002, it was demonstrated an interaction between Vif and APOBEC (1), that inhibits APOBEC (cytidine deaminase) function and at the same time

reduces APOBEC incorporation in the virion.

Earlier studies suggested that Vif functions in viruses producing cells (permissive and non permissive) but its activity influences early stages of viral

infection (2). Other studies also revealed that Vif is necessary to maintain productive viral infection (3).

We have performed several two-hybrid screenings of cDNA libraries (from PBMC, IL-2 stimulated), using Vif1 and Vif2 as baits. In all screenings we have

identified a specific interaction with Histone 3 variant, H3.3, whereas interactions with histones H2A and H2B, and with other HIV proteins, used as

controls, were negative.

CONCLUDING REMARKS

Our results show that Vif interacts specifically with H3.3 and also with H3 (data not shown), which only differs in 2 amino acids. The high levels of homology

obtained between conserved sequences of Vif1 and Vif2 and histone chaperones indicate that Vif could act as a histone chaperon, specific for histone H3.3 and

histone H3.

Although biological relevance of this interaction is still not well defined, if we compare our results with results obtain with others viruses that showed interactions

between viral proteins and histone chaperones that result in viral replication activation (5), we hypothesize that Vif acts as a histone chaperon itself, playing an

important role in transcriptional activation, since H3.3 is an epigenetic positive tag for activation of transcription, in chromatin modulation. Inactivation of H3.3 gene

will clarify the biological meaning of this interaction for HIV replication.

Vif 1

Vif 1(145-192) Vif 1(69-139) Vif 1(9-66)

Vif 2 Vif 2(1-110) Vif 2(110-217) OligoVif 2(139-158)

Histona H3.3

+

+/-

+/- +/-

+

+

+ -

H3.3

H3.3 (1-43)

H3.3 (5´)

H3.3 (3´)

+

+

- + +

+

- +

Vif1 Vif2

49 kDa

~18,7 kDa

Ac α-GST

Ac α-Histone

49 kDa

11 kDa

49 kDa

~15 kDa

A B C

49 kDa

~18,7 kDa

Ac α-GST

Ac α-Histone

49 kDa

11 kDa

49 kDa

~15 kDa

D E F

Using the two-hybrid system we have identified the interacting domain between Vif2 and histone H3.3.

We used different constructs from H3.3, Vif1 and Vif2, as well as, a conserved motif from Vif2 (named OligoVif2) that is localized between 139 and 158 aa residues. As we can observed the domain is localized in the COOH terminal of H3.3 histone and Vif2 protein. For Vif1 and with the constructs that we have used the results are not so conclusives.

HeLa cells were co-transfected with HA-Vif1, HA-Vif2, HA-TE and c-myc histone H3.3. Immunolocalization

was performed at 19h

pos-transfection fixeted cells.

HA-TE (a peroximal enzyme), was used has negative control for HA tag.

On C, F, I its possible to see merge of both tag detections. On panel A and D we see that both Vif1 and Vif2 have nuclear localization, same results were obtain with suspension cell lines, like T cell lines (H9) and macrophagic cell lines (U38).

Lentivirus conserved motif

Vif 2

22% ASF1 anti-silencing function 1 like protein [Cryptosporidium parvum Iowa II]

44% Histone chaperone ASF1, putative [Metarhizium anisopliae ARSEF 23]

88% Histone chaperone RTT106 [Yarrowia lipolytica]

66% Global regulator of transcription [Scheffersomyces stipitis CBS 6054]

C

Vif 1

Lentivirus conserved motif

47% Facilities chromatin transcription comp. subSPT16 [Oryza sativa Japonica ]

47% FACT complex subunit SPT16 [Zea mays]

64% Histone chaperone RTT106 [Yarrowia lipolytica]

35% anti-silencing ASF1-like protein [Cryptosporidium muris RN66]

47% HIRA-interacting protein 3-like [Ailuropoda melanoleuca]

B

Vif 1

Lentivirus conserved motif 83% RNA polymerase II-associated protein 1 Human

66% Hira protein, histone chaperone Hip1 [Schizosaccharomyces pombe 972h-]

100% Anti-silencing protein a-like protein [Leishmania major]

83% Anti-silencing function 1A-like [Oryza sativa Japonica Group]

66% Nucleosome assembly factor [Micromonas sp. RCC29

66% Histone deacetylase 4; Short=HD4

83% Nucleosome assembly protein 1-like protein 3 [Oryza sativa Indica Group]

D

1

HIV-2 Ali MEEGKRWIVV PTWRVPGRME KWHSLVKYLK YRTKDLEKVC YVPHHKVGWA

HIV-1 HxB2 ..NRWQVMI. WQVDRM-.IR T.K...HHMY VSG.ARG-WF .RH.YESPHP

51

HIV-2 Ali WWTCSRVIFP LQGKSHLEIQ AYWNLTPEKG WLSSYAVRIT WYTEKFWTDV

HIV-1 HxB2 RISSEVH.PL GDARLVITT- YWGLH.G.RD .HLGQG.S.E .RKKRYS.Q.

101

HIV-2 Ali TPDCADSLIH GTYFSCFTAG EVRRAIRGEK SLSCCNYPQA HKSQVPSLQF

HIV-1 HxB2 D.EL..Q.Y. LY..DC.SDS AI.K.LL.HI VSPRCE.QAG .-NK.G...Y

151

HIV-2 Ali LALVVVQQNG KPQRNSTTRK QWRRDYRRGL RVARQDSRGL KQRGGESPAP

HIV-1 HxB2 ...AALITPK .IKPPLPSVT KLTE.RWNKP QKTKGHRGSH TMN.H*

201

HIV-2 Ali GAHFPGVAKV LDILA*

Aminoacids in red - conserved motives present in all lentiviruses Ribeiro et al 2005 (4)

A

H

A

-T

E

H

A

-V

if

2

H

A

-V

if

1

c-myc-H3.3 merge

HeLa cells

1. Two-hybrid system mapping domains of Vif/H3.3 interaction

2. Cellular localization of HA-Vif1, HA-Vif2 and cmyc-H3.3 in HeLa transfected cells

4. Homology sequence analysis between conserved amino acid residues from Vif1 and Vif2 proteins and Histone chaperons

3. GST pull-down assay to study Vif/Histone H3.3 interaction

REFERENCES

1-Sheehy, A. M., Gaddis N. C., Choi, J. D., and Malim, M. H. 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646-650

2-Borman, A.M., Quillent, C., Charneau, P., Dauguet,C. and Clavel,F. 1995. Human immunodeffciency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity. J.Virol. 69:2058-2067. 3- Simon JH, Malim MH 1996 The human immunodeficiency virus type 1 Vif protein modulates the postpenetration stability of viral nucleoprotein complexes. J Virol. 70:5297-305

4- Ribeiro, A. C., Maia e Silva, A., Santa-Marta, M., Pombo, A., Moniz-Pereira, J., Goncalves, J., et al. 2005. Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G. J Virol, 79(2): 823-833 5. Placek BJ, Huang J, Kent JR, Dorsey J, Rice L, Fraser NW, Berger SL. 2009. The histone variant H3.3 regulates gene expression during lytic infection with herpes simplex virus type. 1. J Virol. 83:1416-21

Conserved Vif motives, namely the sequence that

encodes the amino acid residues – SLQYLA, present

in all lentiviruses and other conserved motifs

SLVKHHMYVSGKARGWFY (Vif1) and

SLVKYLKRTKDLEKVCY (Vif2) have significant

homology with ASF1 from several eukaryotic organisms.

Homology sequence analysis was performed using BLAST and COBALT program to align multiple protein sequences.

On panel A was shown GST-Vif/H3.3 interaction with Vif from HIV1, as well as, on panel D but in this case the interaction was between Vif from HIV2.

Vif/H4 and H2A interactions were used as negative control (panel B, C, E and F).

GST-Vif (1 and 2) unpurified proteins were incubated with 6 µg of each histone. Proteins were analyzed by Western blot with anti-GST and anti-histone antibodies.

GST pull-down assays have confirm in vitro Vif/H3.3 histone

interaction.

When we use H2A, H2B and H4 in the same assay, we obtain negative

results, suggesting that those histone don’t interact with Vif proteins.