RESUMEN
Influenza viruses remain a major public health concern causing contagious respiratory illnesses that result in around 290,000-650,000 global deaths every year. Their ability to constantly evolve through antigenic shifts and drifts leads to the emergence of newer strains and resistance to existing drugs and vaccines. To combat this, there is a critical need for novel antiviral drugs through the introduction of host-targeted therapeutics. Influenza viruses encode only 14 gene products that get extensively modified through phosphorylation by a diverse array of host kinases. Reversible phosphorylation at serine, threonine, or tyrosine residues dynamically regulates the structure, function, and subcellular localization of viral proteins at different stages of their life cycle. In addition, kinases influence a plethora of signaling pathways that also regulate virus propagation by modulating the host cell environment thus establishing a critical virus-host relationship that is indispensable for executing successful infection. This dependence on host kinases opens up exciting possibilities for developing kinase inhibitors as next-generation anti-influenza therapy. To fully capitalize on this potential, extensive mapping of the influenza virus-host kinase interaction network is essential. The key focus of this review is to outline the molecular mechanisms by which host kinases regulate different steps of the influenza A virus life cycle, starting from attachment-entry to assembly-budding. By assessing the contributions of different host kinases and their specific phosphorylation events during the virus life cycle, we aim to develop a holistic overview of the virus-host kinase interaction network that may shed light on potential targets for novel antiviral interventions.
Asunto(s)
Interacciones Huésped-Patógeno , Gripe Humana , Proteínas Quinasas , Transducción de Señal , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Replicación Viral , Proteínas Quinasas/metabolismo , FosforilaciónRESUMEN
While it is well appreciated that late domains in the viral matrix proteins are crucial to mediate efficient virus budding, little is known about roles of late domains in the viral nucleocapsid proteins. Here, we characterized the functional relevance of a YxxL motif with potential late-domain function in the Ebola virus nucleocapsid protein VP24. Mutations in the YxxL motif had two opposing effects on the functions of VP24. On the one hand, the mutation affected the regulatory function of VP24 in viral RNA transcription and replication, which correlated with an increased incorporation of minigenomes into released transcription- and replication-competent virus-like particles (trVLPs). Consequently, cells infected with those trVLPs showed higher levels of viral transcription. On the other hand, mutations of the YxxL motif greatly impaired the intracellular transport of nucleocapsid-like structures (NCLSs) composed of the viral proteins NP, VP35, and VP24 and the length of released trVLPs. Attempts to rescue recombinant Ebola virus expressing YxxL-deficient VP24 failed, underlining the importance of this motif for the viral life cycle.IMPORTANCE Ebola virus (EBOV) causes a severe fever with high case fatality rates and, so far, no available specific therapy. Understanding the interplay between viral and host proteins is important to identify new therapeutic approaches. VP24 is one of the essential nucleocapsid components and is necessary to regulate viral RNA synthesis and condense viral nucleocapsids before their transport to the plasma membrane. Our functional analyses of the YxxL motif in VP24 suggested that it serves as an interface between nucleocapsid-like structures (NCLSs) and cellular proteins, promoting intracellular transport of NCLSs in an Alix-independent manner. Moreover, the YxxL motif is necessary for the inhibitory function of VP24 in viral RNA synthesis. A failure to rescue EBOV encoding VP24 with a mutated YxxL motif indicated that the integrity of the YxxL motif is essential for EBOV growth. Thus, this motif might represent a potential target for antiviral interference.
Asunto(s)
Ebolavirus/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Línea Celular , Genoma Viral/genética , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/virología , Humanos , Nucleocápside/genética , Nucleocápside/metabolismo , ARN Viral/metabolismo , Virión/genética , Ensamble de Virus/genética , Replicación Viral/fisiologíaRESUMEN
Ebola virus (EBOV) RNA has the potential to form hairpin structures at the transcription start sequence (TSS) and reinitiation sites of internal genes, both on the genomic and antigenomic/mRNA level. Hairpin formation involving the TSS and the spacer sequence between promotor elements (PE) 1 and 2 was suggested to regulate viral transcription. Here, we provide evidence that such RNA structures form during RNA synthesis by the viral polymerase and affect its activity. This was analysed using monocistronic minigenomes carrying hairpin structure variants in the TSS-spacer region that differ in length and stability. Transcription and replication were measured via reporter activity and by qRT-PCR quantification of the distinct viral RNA species. We demonstrate that viral RNA synthesis is remarkably tolerant to spacer extensions of up to ~54 nt, but declines beyond this length limit (~25% residual activity for a 66-nt extension). Minor incremental stabilizations of hairpin structures in the TSS-spacer region and on the mRNA/antigenomic level were found to rapidly abolish viral polymerase activity, which may be exploited for antisense strategies to inhibit viral RNA synthesis. Finally, balanced viral transcription and replication can still occur when any RNA structure formation potential at the TSS is eliminated, provided that hexamer phasing in the promoter region is maintained. Altogether, the findings deepen and refine our insight into structure and length constraints within the EBOV transcription and replication promoter and suggest a remarkable flexibility of the viral polymerase in recognition of PE1 and PE2.
Asunto(s)
Ebolavirus/genética , Estabilidad del ARN/genética , ARN Viral/química , Replicación Viral/genética , Ebolavirus/química , Ebolavirus/fisiología , Genoma Viral/fisiología , Células HEK293 , Fiebre Hemorrágica Ebola/virología , Humanos , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Viral/genética , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Influenza is a negative-sense single-stranded RNA virus with segmented genome. Each segment is encapsidated by a ribonucleoprotein (RNP) complex composed of RNA-dependent RNA polymerase (RdRP) and multiple copies of nucleoprotein (NP). The RNP complex plays a crucial role in viral life cycle, supporting and regulating transcription and replication of viral genome in infected cells. The structural characterization of RdRP and RNP in recent years has shed light on its functions and mechanism of action. In this review, we summarize current understanding on the structure of RNP complex, as well as the structure of each subunit. Crucial functions of RNP are also discussed.