Jeremy A. Bruenn

DNA copies of many non-retroviral RNA virus genes or portions thereof (NIRVs) are present in the nuclear genomes of many eukaryotes. These have often been preserved for millions of years of evolution, suggesting that they play an important cellular function. One possible function is resistance to infection by related viruses. In some cases, this appears to occur through the piRNA system, but in others by way of counterfeit viral proteins encoded by NIRVs. In the fungi, NIRVs may be as long as 1,400 uninterrupted codons. In one such case in the yeast Debaryomyces hansenii, one of these genes provides immunity to a related virus by virtue of expression of a counterfeit viral capsid protein, which interferes with assembly of viral capsids by negative complementation. The widespread occurrence of non-retroviral RNA virus genes in eukaryotes may reflect an underappreciated method of host resistance to infection. This work demonstrates for the first time that an endogenous host protein encoded by a gene that has been naturally acquired from a virus and fixed in a eukaryote can interfere with the replication of a related virus and do so by negative complementation.

The exploration of the evolution of RNA viruses has been aided recently by the discovery of copies of fragments or complete genomes of non-retroviral RNA viruses (Non-retroviral Endogenous RNA Viral Elements, or NERVEs) in many eukaryotic nuclear genomes. Among the most prominent NERVEs are partial copies of the RNA dependent RNA polymerase (RdRP) of the mitoviruses in plant mitochondrial genomes. Mitoviruses are in the family Narnaviridae, which are the simplest viruses, encoding only a single protein (the RdRP) in their unencapsidated viral plus strand. Narnaviruses are known only in fungi, and the origin of plant mitochondrial mitovirus NERVEs appears to be horizontal transfer from plant pathogenic fungi. At least one mitochondrial mitovirus NERVE, but not its nuclear copy, is expressed.

An understanding of the timescale of evolution is critical for comparative virology but remains elusive for many RNA viruses. Age estimates based on mutation rates can severely underestimate divergences for ancient viral genes that are evolving under strong purifying selection. Paleoviral dating, however, can provide minimum age estimates for ancient divergence, but few orthologous paleoviruses are known within clades of extant viruses. For example, ebolaviruses and marburgviruses are well-studied mammalian pathogens, but their comparative biology is difficult to interpret because the existing estimates of divergence are controversial. Here we provide evidence that paleoviral elements of two genes (ebolavirus-like VP35 and NP) in cricetid rodent genomes originated after the divergence of ebolaviruses and cuevaviruses from marburgviruses. We provide evidence of orthology by identifying common paleoviral insertion sites among the rodent genomes. Our findings indicate that ebolaviruses and cuevaviruses have been diverging from marburgviruses since the early Miocene.

Bunyaviridae is a large family of RNA viruses chiefly comprised of vertebrate and plant pathogens. We discovered novel bunyavirids that are approximately equally divergent from each of the five known genera. We characterized novel genome sequences for two bunyavirids, namely, Kigluaik phantom virus (KIGV), from tundra-native phantom midges ( Chaoborus ), and Nome phantom virus (NOMV), from tundra-invading phantom midges, and demonstrated that these bunyavirid-like sequences belong to an infectious virus by passaging KIGV in mosquito cell culture, although the infection does not seem to be well sustained beyond a few passages. Virus and host gene sequences from individuals collected on opposite ends of North America, a region spanning 4,000 km, support a long-term, vertically transmitted infection of KIGV in Chaoborus trivittatus . KIGV-like sequences ranging from single genes to full genomes are present in transcriptomes and genomes of insects belonging to six taxonomic orders, suggesting an ancient association of this clade with insect hosts. In Drosophila , endogenous virus genes have been coopted, forming an orthologous tandem gene family that has been maintained by selection during the radiation of the host genus. Our findings indicate that bunyavirid-host interactions in nonbloodsucking arthropods have been much more extensive than previously thought. IMPORTANCE Very little is known about the viral diversity in polar freshwater ponds, and perhaps less is known about the effects that climate-induced habitat changes in these regions will have on virus-host interactions in the coming years. Our results show that at the tundra-boreal boundary, a hidden viral landscape is being altered as infected boreal phantom midges colonize tundra ponds. Likewise, relatively little is known of the deeper evolutionary history of bunyavirids that has led to the stark lifestyle contrasts between some genera. The discovery of this novel bunyavirid group suggests that ancient and highly divergent bunyavirid lineages remain undetected in nature and may offer fresh insight into host reservoirs, potential sources of emerging disease, and major lifestyle shifts in the evolutionary history of viruses in the family Bunyaviridae .

Background: Plants that utilize the highly efficient C₄ pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C₄ capability and function. In mature C₄ leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO₂ fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C₄ patterns of rbcL gene expression has been an elusive goal for many years. Results: RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C₄ chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C₄ plants. Comparative analysis using maize (C₄) and Arabidopsis (C₃) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions: Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C₄ plants. Evolutionary modification of RLSB expression, from a C₃ “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C₄ plants.

Among eukaryotes with modified nuclear genetic codes, viruses are unknown. However, here we provide evidence of an RNA virus that infects a fungal host (Scheffersomyces segobiensis) with a derived nuclear genetic code where CUG codes for serine. The genomic architecture and phylogeny are consistent with infection by a double-stranded RNA virus of the genus Totivirus. We provide evidence of past or present infection with totiviruses in five species of yeasts with modified genetic codes. All but one of the CUG codons in the viral genome have been eliminated, suggesting that avoidance of the modified codon was important to viral adaptation. Our mass spectroscopy analysis indicates that a congener of the host species has co-opted and expresses a capsid gene from totiviruses as a cellular protein. Viral avoidance of the host’s modified codon and host co-option of a protein from totiviruses suggest that RNA viruses co-evolved with yeasts that underwent a major evolutionary transition from the standard genetic code.

Little is known of the biological significance and evolutionary maintenance of integrated non-retroviral RNA virus genes in eukaryotic host genomes. Here, we isolated novel filovirus-like genes from bat genomes and tested for evolutionary maintenance. We also estimated the age of filovirus VP35-like gene integrations and tested the phylogenetic hypotheses that there is a eutherian mammal clade and a marsupial/ebolavirus/Marburgvirus dichotomy for filoviruses.