RESUMO
A lentiviral vector (LV) pseudotype derived from the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of a murine respirovirus (Sendai virus) facilitates efficient targeting of murine lung in vivo. Since targeting of the human lung will depend upon the availability and distribution of receptors used by F/HN, we investigated transduction of primary human airway cells differentiated at the air-liquid interface (ALI). We observed targeting of human basal, ciliated, goblet, and club cells, and using a combination of sialidase enzymes and lectins, we showed that transduction is dependent on the availability of sialylated glycans, including α2,3 sialylated N-acetyllactosamine (LacNAc). Transduction via F/HN was 300-fold more efficient than another hemagglutinin-based LV pseudotype derived from influenza fowl plague virus (HA Rostock), despite similar efficiency reported in murine airways in vivo. Using specific glycans to inhibit hemagglutination, we showed this could be due to a greater affinity of F/HN for α2,3 sialylated LacNAc. Overall, these results highlight the importance of identifying the receptors used in animal and cell-culture models to predict performance in the human airways. Given the reported prevalence of α2,3 sialylated LacNAc on human pulmonary cells, these results support the suitability of the F/HN pseudotype for human lung gene therapy applications.
RESUMO
Viral tropism, replication, and pathogenesis are determined by multiple interactions between the pathogen and the host. In the case of retroviruses, and in particular, the human immunodeficiency virus, the specific interaction of the envelope protein with the host receptors and co-receptors is essential to gain entry in the cells. After entry, the success of retroviruses to complete their life cycle depends on a complex interplay between the virus and host proteins. Indeed, the cell environment is endowed with a number of factors that actively block distinct stage(s) in the microbial life cycle. Among these restriction factors, Tripartite Motif-5 alpha (TRIM5 alpha) has been extensively studied; however, other TRIM family members have been demonstrated to be anti-retroviral effector proteins. This article reviews, in particular, the current knowledge on the anti-retroviral effects of TRIM5 alpha and TRIM22.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Repressoras/metabolismo , Retroviridae/metabolismo , Replicação Viral , Fatores de Restrição Antivirais , Proteínas de Transporte/química , Humanos , Antígenos de Histocompatibilidade Menor , Proteínas Repressoras/química , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Herpes simplex virus type 1 (HSV-1) and HSV-2 are neurotropic viruses and common human pathogens causing major public health problems such as genital herpes, a sexually transmitted disease also correlated with increased transmission and replication of human immunodeficiency virus type 1 (HIV-1). Therefore, compounds capable of blocking HIV-1, HSV-1, and HSV-2 transmission represent candidate microbicides with a potential added value over that of molecules acting selectively against either infection. We report here that sulfated derivatives of the Escherichia coli K5 polysaccharide, structurally highly similar to heparin and previously shown to inhibit HIV-1 entry and replication in vitro, also exert suppressive activities against both HSV-1 and HSV-2 infections. In particular, the N,O-sulfated [K5-N,OS(H)] and O-sulfated epimerized [Epi-K5-OS(H)] forms inhibited the infection of Vero cells by HSV-1 and -2, with 50% inhibitory concentrations (IC(50)) between 3 +/- 0.05 and 48 +/- 27 nM, and were not toxic to the cells at concentrations as high as 5 muM. These compounds impaired the early steps of HSV-1 and HSV-2 virion attachment and entry into host cells and reduced the cell-to-cell spread of HSV-2. Since K5-N,OS(H) and Epi-K5-OS(H) also inhibit HIV-1 infection, they may represent valid candidates for development as topical microbicides preventing sexual transmission of HIV-1, HSV-1, and HSV-2.
Assuntos
Cápsulas Bacterianas/farmacologia , Células Epiteliais/virologia , Escherichia coli/química , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Sulfatos/farmacologia , Animais , Cápsulas Bacterianas/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/patogenicidade , Humanos , Recombinação Genética , Sulfatos/metabolismo , Células VeroRESUMO
Quasispecies composition and tissue distribution of feline coronaviruses (FCoVs) were studied in naturally infected cats. The genomic complexity of FCoVs was investigated using single-strand conformational polymorphism (SSCP) analysis of N and ORF7b amplicons, and the evolutionary process was investigated by sequence-based phylogenetic analysis. SSCP analysis showed high heterogeneity of the FCoV genome which was correlated with the seriousness of the clinical form. The two genomic regions analysed showed different levels of variation; the N region demonstrated significant heterogeneity as compared to ORF7b. Phylogenetic analysis of the nucleotide sequences showed the clear separation of sequences analysed on the basis of virulence and geographical origin. A maximum likelihood analysis of N and ORF7b data sets showed a situation of strong heterogeneity for the N region.
Assuntos
Infecções por Coronavirus/veterinária , Coronavirus Felino/genética , Coronavirus Felino/isolamento & purificação , Genoma Viral , Filogenia , Animais , Gatos/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Coronavirus Felino/classificação , Peritonite Infecciosa Felina/epidemiologia , Peritonite Infecciosa Felina/virologia , Genes Virais/genética , Genética Populacional , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNARESUMO
OBJECTIVE: Functional polarization of human monocyte-derived macrophages (MDMs) into M1 cells leads to inhibition of R5 HIV-1 replication and viral DNA synthesis in comparison to control, unpolarized cells together with CD4 downregulation from the cell surface and upregulation of CCR5-binding chemokine secretion. We here investigated whether a postentry restriction of virus replication is also induced by M1 polarization of MDM. DESIGN: MDM were first polarized to M1 cells by 18 h stimulation with interferon-[gamma] and tumor necrosis factor-[alpha]; the cytokines were then removed and the cells were infected with vesicular stomatitis virus G-protein pseudotyped enhanced green fluorescence protein HIV-1 (HIV-GFP) generating a single-round infection cycle. METHODS: HIV-1 expression was monitored in terms of eGFP expression by fluorescence activated cell sorter (FACS) analysis and real-time PCR analysis of total HIV-1 gag DNA, 2-long terminal repeat DNA, proviral DNA, and multiply spliced RNA transcripts. Expression of apolipopoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), and APOBEC3A was tested by western blotting and FACS analysis. RESULTS: Inhibition of HIV-GFP expression was observed in M1-MDM along with impaired viral DNA synthesis, delayed proviral integration, and reduced proviral transcription. Although APOBEC3G levels were similar in M1 and unpolarized MDM, APOBEC 3A was selectively expressed only by M1 cells. CONCLUSION: M1 polarization of in-vitro differentiated primary MDM determines a transient, but profound restriction of HIV-1 replication affecting multiple (entry and postentry) steps in the virus life cycle likely involving the upregulated expression of APOBEC3A.