RESUMEN

Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. IMPORTANCE: Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.

Asunto(s)

Culex/virología , Culicidae/genética , Flavivirus/genética , Interferencia de ARN/fisiología , ARN Viral/genética , Fiebre del Nilo Occidental/transmisión , Virus del Nilo Occidental/genética , Regiones no Traducidas 3'/genética , Animales , Línea Celular , Chlorocebus aethiops , Culex/genética , Culicidae/virología , Virus del Dengue/genética , Insectos Vectores/genética , ARN Interferente Pequeño/genética , Células Vero , Fiebre del Nilo Occidental/virología , Virus de la Fiebre Amarilla/genética , Virus Zika/genética , Infección por el Virus Zika/transmisión , Infección por el Virus Zika/virología

RESUMEN

We demonstrated previously that CD45RA(+) CD4(+) T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO(+) CD4(+) T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA(+) and CD45RO(+) CD4(+) cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO(+) CD4(+) cells. SI variants seemed to evolve in CD45RO(+) CD4(+) cells, but, in time, SI HIV-1 infection of CD45RA(+) CD4(+) cells equaled infection of CD45RO(+) CD4(+) cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA(+) and CD45RO(+) cells, which allowed us to isolate virus from purified CCR5(+) CXCR4(-) and CCR5(-) CXCR4(+) CD4(+) cells. The CCR5(+) subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4(+) subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways.

Asunto(s)

Linfocitos T CD4-Positivos/virología , Evolución Molecular , Células Gigantes/virología , Infecciones por VIH/virología , VIH-1/genética , Receptores del VIH/genética , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/clasificación , Variación Genética , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/patogenicidad , Humanos , Antígenos Comunes de Leucocito , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fenotipo , Filogenia , Receptores CCR5/genética , Receptores CXCR4/genética , Homología de Secuencia de Aminoácido , Subgrupos de Linfocitos T/virología

RESUMEN

Fifty percent of individuals infected with human immunodeficiency virus type 1 (HIV-1) progress to AIDS in the presence of only non-syncytium-inducing (NSI) variants. These rapidly replicating NSI isolates are associated with a high viral load. The question of whether disease progression in the absence of syncytium-inducing (SI) HIV-1 variants is associated with an expansion of the coreceptor repertoire of NSI HIV-1 variants was studied. Biological HIV-1 clones were isolated both early and late in infection from progressors and long-term survivors with wild-type or mutant CCR5 or CCR2b genotypes and analyzed for their capacity to use CCR1, CCR2b, CCR3, CCR5, and CXCR4 on U87 cells coexpressing CD4. All HIV-1 clones were restricted to the use of CCR5. Absent replication of all HIV-1 clones in peripheral blood mononuclear cells from a CCR5 Delta32 homozygous blood donor confirmed this result. These findings indicate that an expanded coreceptor repertoire of HIV-1 is not a prerequisite for a progressive clinical course of HIV-1 infection.

Asunto(s)

Infecciones por VIH/inmunología , Infecciones por VIH/fisiopatología , VIH-1 , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Antígenos CD4/genética , Antígenos CD4/inmunología , Línea Celular , Progresión de la Enfermedad , Estudios de Seguimiento , Variación Genética , Genotipo , Infecciones por VIH/genética , VIH-1/genética , Homosexualidad Masculina , Humanos , Estudios Longitudinales , Masculino , Valor Predictivo de las Pruebas , Pronóstico , Receptores CCR1 , Receptores CCR2 , Receptores CCR3 , Receptores CCR5/genética , Receptores CXCR4/genética , Receptores de Citocinas/genética , Sobrevivientes , Linfocitos T/inmunología , Factores de Tiempo , Transfección , Carga Viral

RESUMEN

Heterozygosity for a 32-bp deletion in the CCR5 gene (CCR5 Delta32), which encodes the coreceptor for macrophage-tropic non-syncytium-inducing (NSI) human immunodeficiency virus type 1 (HIV-1) variants, results in a lower CCR5 expression and reduced NSI HIV-1 replication. Because infection of macrophages and microglial cells by NSI HIV-1 is considered to be instrumental for the development of AIDS dementia complex (ADC), we studied whether the CCR5 Delta32 heterozygous genotype correlated with a reduced frequency of ADC. Two (4.1%) of 49 patients with ADC versus 27 (14. 5%) of 186 AIDS patients without ADC were heterozygous for CCR5 Delta32 (P=.05). In contrast, a point mutation in the first transmembrane domain of CCR2 (CCR2 64I) did not show this protective effect (P=.57). The reduced prevalence of the CCR5 Delta32 allele among patients with ADC may indicate a reduced or absent reservoir of macrophage-tropic NSI HIV-1 in the brain of CCR5 Delta32 heterozygotes.

Asunto(s)

Complejo SIDA Demencia/genética , Síndrome de Inmunodeficiencia Adquirida/genética , Heterocigoto , Receptores CCR5/genética , Complejo SIDA Demencia/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Encéfalo/virología , Estudios de Casos y Controles , Genotipo , VIH-1/aislamiento & purificación , VIH-1/fisiología , Homocigoto , Humanos , Macrófagos/virología , Eliminación de Secuencia , Replicación Viral

RESUMEN

The effect of a valine to isoleucine switch in the CCR2 first transmembrane domain (CCR2 64I) on the clinical course of human immunodeficiency virus type 1 (HIV-1) infection was analyzed in relation to the presence or absence of syncytium-inducing (SI) HIV-1 variants. Compared with persons with a wild-type genotype for CCR2 and CCR5, subjects with a CCR2-64I/+ or 64I/64I (but CCR5 wild-type homozygous genotype) had significantly delayed disease progression (relative hazard, 0.66; 95% confidence interval, 0.44-0.99) with a 1. 5-fold slower CD4 T lymphocyte decline and a 1.2-fold lower RNA virus load. The delay in disease progression was more pronounced when only non-SI (NSI) HIV-1 variants were present and was not observed after conversion to SI HIV-1 in CCR2-64I/+ persons. In CCR2-64I/+ subjects, a higher conversion rate to and a higher prevalence of SI HIV-1 was observed. These findings suggest that the mechanism of action of the CCR2 polymorphism is mediated via CCR5-restricted NSI HIV-1 variants.

Asunto(s)

Variación Genética , Seropositividad para VIH/genética , VIH-1/genética , Mutación Puntual , Receptores de Quimiocina/genética , Receptores de Citocinas/genética , Células Cultivadas , Intervalos de Confianza , Progresión de la Enfermedad , Genotipo , Células Gigantes , Seropositividad para VIH/inmunología , Seropositividad para VIH/fisiopatología , VIH-1/patogenicidad , Homosexualidad Masculina , Humanos , Isoleucina , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Masculino , Modelos de Riesgos Proporcionales , Receptores CCR2 , Receptores CCR5/genética , Factores de Tiempo , Valina

RESUMEN

BACKGROUND: A G-to-A transition in the 3' untranslated region (UTR) of stromal cell-derived factor (SDF)-1 gene (SDF1-3'A) has recently been described, which in the homozygous state was associated with delayed disease progression. OBJECTIVE: To analyse the effect of the SDF-1 polymorphism on AIDS-free survival and survival after AIDS diagnosis, also in relation to viral phenotype. DESIGN: Retrospective longitudinal study among 344 homosexual HIV-1-infected men. RESULTS: A more rapid progression to AIDS (Centers for Disease Control and Prevention 1993 definition) was observed in SDF1-3'A/3'A subjects than in wild-type (SDF1-wt/wt) subjects (relative hazard, 1.75; P = 0.07). Using death as an endpoint, accelerated progression was no longer observed (relative hazard, 0.93; P = 0.84), suggesting a late protective effect of the SDF1-3'A/3'A genotype. Indeed, survival after AIDS diagnosis was significantly delayed in SDF1-3'A/3'A subjects (relative hazard, 0.40; P = 0.02). No effect of the SDF1-3'A/wt genotype on disease progression was observed. Interestingly, a higher frequency of Kaposi's sarcoma was observed as the AIDS-defining event among SDF1-3'A/3'A (40.0%) and SDF1-3'A/wt (30.6%) subjects than in SDF1-wt/wt subjects (17.0%). At the end of the study the total frequency of syncytium-inducing (SI) HIV-1 variants was lower in SDF1-3'A/3'A subjects (22.2%) than in SDF1-3'A/wt (32.5%) and SDF1-wt/wt subjects (40.5%), although not significantly. SDF-1 genotype did not influence the rate of evolution to SI HIV-1. Progression to AIDS after the emergence of SI HIV-1 was accelerated in SDF1-3'A/3'A subjects compared with the SDF1-wt/wt genotypic group (relative hazard, 4.04; P = 0.06). CONCLUSIONS: In our study group, homozygosity for a G-to-A transition in the 3' UTR of SDF-1 is associated with an accelerated progression to AIDS but a subsequent prolonged survival after AIDS diagnosis.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/genética , Quimiocinas CXC/genética , VIH-1 , Células del Estroma , Síndrome de Inmunodeficiencia Adquirida/mortalidad , Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Síndrome de Inmunodeficiencia Adquirida/virología , Quimiocina CXCL12 , Progresión de la Enfermedad , Estudios de Seguimiento , Variación Genética , Genotipo , VIH-1/genética , Homosexualidad Masculina , Humanos , Estudios Longitudinales , Masculino , Fenotipo , Estudios Prospectivos , Estudios Retrospectivos , Sobrevivientes