AKAP-Lbc is a Rho-activating guanine nucleotide exchange factor (RhoGEF) important in heart development and pro-fibrotic signaling in cardiomyocytes. Heterotrimeric G proteins of the G12/13 subfamily, comprising Gα12 and Gα13, are well characterized as stimulating a specialized group of RhoGEFs through interaction with their RGS-homology (RH) domain. Despite lacking an RH domain, AKAP-Lbc is bound by Gα12 through an unknown mechanism to activate Rho signaling. We identified a Gα12-binding region near the C-terminus of AKAP-Lbc, closely homologous to a region of p114RhoGEF that we also discovered to interact with Gα12. This binding mechanism is distinct from the well-studied interface between RH-RhoGEFs and G12/13 α subunits, as demonstrated by Gα12 mutants selectively impaired in binding either this AKAP-Lbc/p114RhoGEF region or RH-RhoGEFs. AKAP-Lbc and p114RhoGEF showed high specificity for binding Gα12 in comparison to Gα13, and experiments using chimeric G12/13 α subunits mapped determinants of this selectivity to the N-terminal region of Gα12. In cultured cells expressing constitutively GDP-bound Gα12 or Gα13, the Gα12 construct was more potent in exerting a dominant-negative effect on serum-mediated signaling to p114RhoGEF, demonstrating coupling of these signaling proteins in a cellular pathway. In addition, charge-reversal of conserved residues in AKAP-Lbc and p114RhoGEF disrupted Gα12 binding for both proteins, suggesting they harbor a common structural mechanism for interaction with this α subunit. Our results provide the first evidence of p114RhoGEF as a Gα12 signaling effector, and define a novel region conserved between AKAP-Lbc and p114RhoGEF that allows Gα12 signaling input to these non-RH RhoGEFs.

Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

Orthohantaviruses are rodent-borne, negative-sense RNA viruses that are capable of causing severe vascular disease in humans. Over the course of viral evolution, these viruses have tailored their replication cycles in such a way as to avoid and/or antagonize host innate immune responses. In the rodent reservoir, this results in life long asymptomatic infections. However, in hosts other than its co-evolved reservoir, the mechanisms for subduing the innate immune response may be less efficient or absent, potentially leading to disease and/or viral clearance. In the case of human orthohantavirus infection, the interaction of the innate immune response with viral replication is thought to give rise to severe vascular disease. The orthohantavirus field has made significant advancements in understanding how these viruses replicate and interact with host innate immune responses since their identification by Dr. Ho Wang Lee and colleagues in 1976. Therefore, the purpose of this review, as part of this special issue dedicated to Dr. Lee, was to summarize the current knowledge of orthohantavirus replication, how viral replication activates innate immunity, and how the host antiviral response, in turn, impacts viral replication.

Alphaviruses are positive-sense RNA arboviruses that are capable of causing severe disease in otherwise healthy individuals. There are many aspects of viral infection that determine pathogenesis and major efforts regarding the identification and characterization of virulence determinants have largely focused on the roles of the nonstructural and structural proteins. Nonetheless, the viral RNAs of the alphaviruses themselves play important roles in regard to virulence and pathogenesis. In particular, many sequences and secondary structures within the viral RNAs play an important part in the development of disease and may be considered important determinants of virulence. In this review article, we summarize the known RNA-based virulence traits and host:RNA interactions that influence alphaviral pathogenesis for each of the viral RNA species produced during infection. Overall, the viral RNAs produced during infection are important contributors to alphaviral pathogenesis and more research is needed to fully understand how each RNA species impacts the host response to infection as well as the development of disease.

Alphaviruses are positive-sense RNA arboviruses that are capable of causing severe disease in otherwise healthy individuals. There are many aspects of viral infection that determine pathogenesis and major efforts regarding the identification and characterization of virulence determinants have largely focused on the roles of the nonstructural and structural proteins. Nonetheless, the viral RNAs of the alphaviruses themselves play important roles in regard to virulence and pathogenesis. In particular, many sequences and secondary structures within the viral RNAs play an important part in the development of disease and may be considered important determinants of virulence. In this review article, we summarize the known RNA-based virulence traits and host:RNA interactions that influence alphaviral pathogenesis for each of the viral RNA species produced during infection. Overall, the viral RNAs produced during infection are important contributors to alphaviral pathogenesis and more research is needed to fully understand how each RNA species impacts the host response to infection as well as the development of disease.

Hantaviruses are zoonotic, tri-segmented, negative-sense RNA viruses and a significant public health threat. Viral pathogenesis varies between host species, with rodent reservoir infection being asymptomatic and human infection resulting in severe, immune-mediated disease. Viral pathogenesis is highly dependent on virus replication efficiency since it affects the virus’s ability to evade detection and determines the magnitude of the host immune response. However, the molecular replication kinetics for hantaviruses remain poorly defined. Therefore, we developed a sense- and segment-specific quantitative real-time PCR assay and an SYBR-based RT-qPCR assay, allowing us to quantify both negative-sense genome levels and total viral RNA synthesis of the small (S), medium (M), and large (L) segments of Seoul virus (SEOV). We then measured total viral RNA and genome accumulation in reservoir rat endothelial cells (RLMVEC), non-reservoir human endothelial cells (HUVEC-C), and Vero E6 epithelial cells. We also measured the ratio of each segment released into the culture supernatant, approximating the relative packaging efficiency. We found that, while the magnitude of viral RNA differed, RNA replication kinetics were largely similar between reservoir and non-reservoir endothelial cells. However, replication and release kinetics differed between infection of endothelial and Vero cells. We also found that the S, M, and L segments were not equally abundant during viral infection or release but instead followed a trend of M>L>S. Overall, this study validates two RT-qPCR assays to measure SEOV RNA, details the accumulation and release of each viral segment and demonstrates the impact of host cell type on hantavirus replication.