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J Miguel

Azevedo Pereira
FFUL

HIV - estrutura,
organizao gentica e ciclo de
replicao

HIV-2ALI; Azevedo-Pereira et al.


unpublished results

Jos Miguel Azevedo Pereira


CPM-URIA-FFUL
e-mail: miguel.pereira@ff.ul.pt
URL: http://web.mac.com/jmiguelap/Entrada_geral/Entrada.html

Famlia Retroviridae
Sub-famlia

J Miguel
Azevedo Pereira
FFUL

Gnero

Exemplos de espcies

Alpharetrovirus

Avian leukosis virus; Rous sarcoma virus

Betaretrovirus

Mouse mammary tumor virus; Mason-Pfizer


monkey virus

Gammaretrovirus Murine leukemia virus; Feline leukemia virus;

Orthoretrovirinae

Spumaretrovirinae

Deltaretrovirus

Bovine leukemia virus; Human Tlymphotropic virus 1; Human T-lymphotropic


virus 2

Epsilonretrovirus

Walleye dermal sarcoma virus

Lentivirus

Human immunodeficiency virus 1; Human


immunodeficiency virus 2; Simian
immunodeficiency virus; Bovine
immunodeficiency virus; Feline
immunodeficiency virus; Visna/maedi virus

Spumavirus

Simian foamy virus 1, Simian foamy virus 3

Origem do HIV

A SIDA uma zoonose transmitida do


macaco ao Homem (SIV)

HIV-1 ter sido transmitido inicialmente por


volta de 1930 15

HIV-2 ter sido transmitido por volta de


1940 16

Podem estar a ocorrer episdios zoonticos


no presente (Peeters et al. 2002)

J Miguel
Azevedo Pereira
FFUL

Caractersticas do HIV

Vrus com invlucro


Dois tipos: HIV-1 e HIV-2
Agente causal da SIDA
Genoma RNA (duas cadeias)
100 nm de dimetro
Nucleocpside cnica

J Miguel
Azevedo Pereira
FFUL

Estrutura do HIV

J Miguel
Azevedo Pereira
FFUL

J Miguel
Azevedo Pereira
FFUL

Protenas do gene gag

Sintetizadas como uma poliprotena precursora que clivada dando


origem s protenas:

Protena da matriz (MA), localizada entre a cpside e o invlucro viral


Protena da cpside (CA), que forma o core viral
Protena da nucleocpside (NC), que se encontra intimamente ligada ao
RNA genmico

J Miguel
Azevedo Pereira
FFUL

Protenas do gene pol

Sintetizadas como uma poliprotena precursora (Gag-Pol)


que clivada dando origem s protenas:

Protease (PR)
Transcriptase reversa (TR)
Integrase (IN)

J Miguel
Azevedo Pereira
FFUL

Glicoprotenas do gene env

J Miguel
Azevedo Pereira
FFUL

O gene env codifica duas glicoprotenas: a glicoprotena de


superfcie (SU ou gp120) e a glicoprotena transmembranar
(TM ou gp41)

Sintetizadas sob a forma de uma protena precursora


(gp160) altamente glicosilada que clivada por aco de
uma protease celular

Glicoprotena de superfcie (SU)

Responsvel pela ligao aos receptores celulares (CD4 e


receptor das quimiocinas) que culmina com a fuso do vrus
com a clula

Principal determinante antignico


Elevada variabilidade gentica
Adaptado de Doms & Moore 2000

J Miguel
Azevedo Pereira
FFUL

Glicoprotena transmembranar (TM)

Relativamente hidrofbica, parte da sua molcula est inserida na bicamada


lipdica do invlucro viral

Est ligada SU por ligaes no covalentes (heterodmeros SU-TM)

Envolvida no processo de fuso vrus-clula (pptido de fuso)

A sua regio N-terminal, externa ao invlucro viral, est aparentemente


envolvida na trimerizao dos heterodmeros SU-TM

Adaptado de Doms & Moore 2000

J Miguel
Azevedo Pereira
FFUL

Protenas reguladoras

J Miguel
Azevedo Pereira
FFUL

J Miguel
Azevedo Pereira
FFUL

Protena

Tipo

Funes

Tat

Precoce; 14 KDa; localizao


nuclear

Activao da transcrio

Rev

Precoce; 19 KDa; localizao


nuclear

Nef
Vif
Vpr
Vpu
Vpx

Processamento e transporte
dos mRNA
Precoce; 27 KDa; presente no Degradao do CD4; apoptose;
citoplasma e no virio
estimulao dos Ly T; MHC I
Eficincia da retrotranscrio;
Tardia; 23 KDa; presente no
inibidor da protena celular
citoplasma e no virio
Apobec3G
Tardia; 14 KDa; presente no
Entrada do PIC no ncleo;
citoplasma e no virio
bloqueio na fase G2
Tardia; 16 KDa; aparelho de Degradao do CD4; aumento
Golgi e RE
da libertao de viries
Tardia; 12 KDa; s presente no Desconhecida; sinergia com a
HIV-2 e certos SIV
Vpr(?)

Ciclo de replicao

J Miguel
Azevedo Pereira
FFUL

Pathogenesis
of HIV infection:
Virologic
a s p e c t s

Jos Miguel
Azevedo Pereira
URIA-CPM-FFUL

HIV-2ALI (Azevedo-Pereira, unpublished results)

www.pfizerpro.com
J Miguel Azevedo Pereira - URIA

The HIV...

http://en.wikipedia.org
J Miguel Azevedo Pereira - URIA

www.pfizerpro.com
J Miguel Azevedo Pereira - URIA

HIV Env glycoproteins


www.mcld.co.uk/

J Miguel Azevedo Pereira - URIA

Viral determinants for HIV tropism


HIV-1

J Miguel Azevedo Pereira - URIA

The cell...

J Miguel Azevedo Pereira - URIA

www.cdc.gov/

www.pfizerpro.com
J Miguel Azevedo Pereira - URIA

Doms. Top HIV Med, 2004


www.pfizerpro.com
J Miguel Azevedo Pereira - URIA

Chemokine receptors as
HIV coreceptors
CCR1

CCR2b

CCR3

CCR4

CCR5

CXCR2

CXCR4

CXCR5

CXCR6

CX3CR1

GPR-1

GPR-15

Apj

ChemR23

RDC1

CCR8

CCR9

BLTR

US28

Azevedo-Pereira et al. Curr. HIV Res, 2005


J Miguel Azevedo Pereira - URIA

For HIV-1...
CCR5 (R5)

CXCR4 (X4)

CCR5 + CXCR4 (R5X4)


...

J Miguel Azevedo Pereira - URIA

For HIV-2...
CCR5

CXCR4

+
CCR1, CCR2b, CCR3, CCR4, CCR8,
CXCR2, CXCR5, GPR-1, RDC-I

(Bron 1997, Guillon 1998, McKnight 1998)

Do not use neither CCR5 nor CXCR4


(Sol 1997, Azevedo-Pereira 2003)

Infection of target cells in the absence of CD4


(Reeves 1999, Azevedo-Pereira 2003)

J Miguel Azevedo Pereira - URIA

CD4-independent infection

Reeves and Doms. J Gen Virol 2002

J Miguel Azevedo Pereira - URIA

Consequences of HIV
entry
Delivery of HIV genetic material into host cell

J Miguel Azevedo Pereira - URIA

Productive cycle vs. abortive cycle

J Miguel Azevedo Pereira - URIA

Productive cycle vs. abortive cycle

Full permissive cell


Stevenson. Nat Med 2003

Must be activated in a
short time frame in order
to integration occurs
J Miguel Azevedo Pereira - URIA

Consequences of HIV
entry
Delivery of HIV genetic material into host cell
Cellular receptors engagement by Env
glycoproteins triggers the activation of signal
transducing pathways

J Miguel Azevedo Pereira - URIA

Receptor signaling
Not required for receptor function in HIV fusion step
(Amara et al. J Virol 2003)

Directly influence the intracellular viral life cycle after


entry
Increasing the activation state of target cells (Davis et al. J Exp Med
1997)

Regulating cell growth and differentiation (Arthos et al. J Virol 2000)


Influencing cell survival - apoptosis (Cicala et al. PNAS 2000)
Inducing modifications in cytoskeletal structure (Sasaki et al. BBRC
2004)

J Miguel Azevedo Pereira - URIA

R5 virus Memory CD4+


T-cells
DCs
Macrophages
X4 virus Naive
CD4+
T-cells

J Miguel Azevedo Pereira - URIA

CXCR4 (X4) strains


Increased replication kinetics
More cytopathic phenotype
Ability to infect an expanded target cell
population repertoire in vivo, including
timocytes
In HIV-1, they arise as a consequence of few
amino acid changes in V3 region of SU
glycoprotein
J Miguel Azevedo Pereira - URIA

Why X4 viruses only predominate


(...) in late disease stages?

Regoes et al. Trends in Microb 2005

J Miguel Azevedo Pereira - URIA

CCR5 dependence of early


events during transmission
Epithelial cells express mainly CCR5 and not
CXCR4 (Meng et al. Nat Med 2002)
Dendritic cells bind R5 virus preferentially (GraneliPiperno et al. J Virol 1998; Reece et al. J Exp Med 1998)

High-levels of SDF-1 (a CXCR4-blocking


ligand) is expressed by mucosal surfaces (Agace et
al. Curr Biol 2000)

J Miguel Azevedo Pereira - URIA

However...
Predominance of R5 virus early in infection
is independent of the route of transmission
(parenteral=mucosal)

J Miguel Azevedo Pereira - URIA

Replicative
advantage of
R5 virus

Less cytopathic =
longer life-span of
infected cells
And... Or...

Host immune response inhibits more efficiently the


replication of X4 virus than R5 variants

J Miguel Azevedo Pereira - URIA

R5 to X4 switch - more constrains...

Regoes et al. Trends in Microb 2005


J Miguel Azevedo Pereira - URIA

Regoes et al. Trends in Microb 2005

and more...

J Miguel Azevedo Pereira - URIA

Selection by target cells


Replicative disadvantage
Host immune control
Selection of mutants
Viral fitness of intermediates

J Miguel Azevedo Pereira - URIA

HIV-2 pathogenesis
Lower plasma viremia
Lower transmission rates - restricted
geographic distribution
Prolonged asymptomatic period
Lower CD4+ T-lymphocyte depletion
Far more sensitive to neutralizing antibodies
J Miguel Azevedo Pereira - URIA

Broader
coreceptor usage

CD4-independent
infection

More relaxed conformation of HIV-2


Env glycoproteins
Exposure of critical epitopes in coreceptor
binding site
Induction of efficient
neutralizing antibodies
J Miguel Azevedo Pereira - URIA

Broader coreceptor usage

Infection of inappropriate cell populations

Replication cycle aborted in some intracellular step

Lower replication lower plasma viral load


J Miguel Azevedo Pereira - URIA

Inappropriate engagement of cellular receptors


Inefficient signal transduction
Inefficient cell
activation

Lower apoptosis
induction

Lower virus
production

Minor CD4 ly
depletion

Slower disease
progression
J Miguel Azevedo Pereira - URIA

Diagnstico da infeco
Mtodos indirectos ou serolgicos: deteco de anticorpos antiHIV no soro ou plasma (rastreio e confirmao)
Indivduo com anticorpos = indivduo infectado (excepto se a idade for < 18
meses)

Mtodos directos:
Deteco das partculas virais (cultura viral) no sangue (CMSP)
Deteco de antignio viral (Ag p24) no soro/plasma
Deteco do DNA proviral nas CMSP

Determinao da carga viral: prognstico da infeco e


monitorizao da teraputica (plasma)
J Miguel Azevedo Pereira
FFUL

Testes Imunoenzimticos
Antignios: Lisado viral total; protenas recombinantes;
pptidos sintticos
Os mais recentes (4 gerao) incluem Ag do HIV-1
(todos os subtipos; incluindo o subtipo O) e HIV-2 e
anticorpo monoclonal para o Ag p24
Requerem confirmao dos resultados positivos
(Western-blot)
J Miguel Azevedo Pereira
FFUL

Critrios de classificao do
WB
Positivo: anticorpos para, pelo
menos, dois dos antignios
codificados pelo gene env (gp160,
gp120, gp41; gp140, gp105, gp36)
Negativo: ausncia de anticorpos
para qualquer dos antignios virais
Indeterminado: anticorpos para os
antignios virais, excepto os
codificados pelo gene env

J Miguel Azevedo Pereira


FFUL