Isolation and Identification of Inter-Species Enterovirus Recombinant Genomes

Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.

Covid-19 Vaccination: A Bird’s Eye View

Covid-19 pandemic plagued this world since the beginning of 2020 AD. It is caused by a new positive-strand RNA virus of coronaviridae family [1]. It causes Coronavirus disease 2019 (hence the name COVID-19). It is a contagious disease predominantly causes severe acute respiratory syndrome, hence the name SARS-CoV-2. It started from Wuhan, China, in December 2019. Since then, it has spread globally. It is reported to be a new virus therefore it’s properties, pathogenesis, virulence, immunogenicity, variants, and how will host body will react to this virus was unknown. Despite of 22 months since this virus started to spread worldwide, researchers and clinicians continued to learn about it on daily basis. Newer information about it poured in daily in scientific journals as well as in print / electronic media. Mostly, newer information continued to negate earlier information. Social media disinformation continued to confuse the masses.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle

A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes.

Ultrastructural Features of Membranous Replication Organelles Induced by Positive-Stranded RNA Viruses

All intracellular pathogens critically depend on host cell organelles and metabolites for successful infection and replication. One hallmark of positive-strand RNA viruses is to induce alterations of the (endo)membrane system in order to shield their double-stranded RNA replication intermediates from detection by the host cell’s surveillance systems. This spatial seclusion also allows for accruing host and viral factors and building blocks required for efficient replication of the genome and prevents access of antiviral effectors. Even though the principle is iterated by almost all positive-strand RNA viruses infecting plants and animals, the specific structure and the organellar source of membranes differs. Here, we discuss the characteristic ultrastructural features of the virus-induced membranous replication organelles in plant and animal cells and the scientific progress gained by advanced microscopy methods.

Tombusviruses target a major crossroad in the endocytic and recycling pathways via co-opting Rab7 small GTPase

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles, called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in relocalization of Rab7 into the large VROs. Similar to depletion of Rab7, deletion of either MON1 or CCZ1 heterodymeric GEFs (guanine nucleotide exchange factors) of Rab7, inhibited TBSV repRNA replication in yeast. This suggests that the activated Rab7 has pro-viral functions. We show that the pro-viral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting Rab7 into VROs results in delivery of several retromer cargos with pro-viral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 (phosphatidylinositol 4-kinase) and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. Importance: Replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, the authors discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has pro-viral functions through facilitating the delivery of co-opted retromer complex, sorting nexin-BAR proteins and lipid enzymes into VROs to create optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.

nsP4 is a major determinant of alphavirus replicase activity and template selectivity

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the non-structural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1-nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis -active sequences. Here we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123 while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. Importance. A key element of every virus with an RNA genome is the RNA replicase. Understanding the principles of RNA replicase formation and functioning is therefore crucial for understanding and responding to the emergence of new viruses. Reconstruction of the replicases of nine alphaviruses from nsP4 and P123 polyproteins revealed that the nsP4 of the majority of alphaviruses, including the mosquito-specific Eilat virus, could form a functional replicase with P123 originating from a different virus, and the corresponding chimeric viruses were replication-competent. nsP4 also had an evident role in determining the template RNA preference and the efficiency of RNA synthesis. The revealed broad picture of the compatibility of the replicase components of alphaviruses is important for understanding the formation and functioning of the alphavirus RNA replicase and highlights the possibilities for recombination between different alphavirus species.