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Sin Nombre orthohantavirus (SNV), a negative-sense, single-stranded RNA virus that is carried and transmitted by the North American deer mouse Peromyscus maniculatus, can cause infection in humans through inhalation of aerosolized excreta from infected rodents. This infection can lead to hantavirus cardiopulmonary syndrome (HCPS), which has a ∼36% case-fatality rate. We used reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to confirm SNV infection in a patient and identified SNV in lung tissue in wild-caught rodents from potential sites of exposure. Using viral whole-genome sequencing (WGS), we identified the likely site of transmission and discovered SNV in multiple rodent species not previously known to carry the virus. Here we report, for the first time, the use of SNV WGS to pinpoint a likely site of human infection and identify SNV simultaneously in multiple rodent species in an area of known host-to-human transmission. These results will impact epidemiology and infection control for hantaviruses by tracing zoonotic transmission and investigating possible novel host reservoirs. Importance Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nombre orthohantavirus (SNV) is the main cause of hantavirus disease in North America. In this study, we sequenced SNV from an infected patient and wild-caught rodents to trace location of infection. We also discovered SNV in rodent species not previously known to carry SNV. These studies demonstrate for the first time the use of virus sequencing to trace transmission of SNV, and describe infection in novel rodent species.

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5′ termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5′-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5′-to-3′ exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5′-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5′ access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5′ terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5′ access, we have termed this 5′-terminal structure an Xrn-evading RNA, or xeRNA. These and other findings demonstrate that the 5′UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5′ terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5′ capped. Here, we explored how a species in the type genus Tombusvirus protects its genomic 5′ end from cellular nuclease attack. Our results revealed that the 5′-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5′-to-3′ exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5′-triphosphorylated RNA termini into 5′-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5′-end-attacking cellular enzymes.

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to 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.

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wild-type viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. In this study, we analyzed in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wild-type population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionarily conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications. IMPORTANCE The use of defective viruses that interfere with the replication of their infectious parent after coinfecting the same cells—a therapeutic strategy known as viral interference—has recently generated a lot of interest. The CRISPR-based system that we recently reported for herpesviruses represents a novel interfering strategy that causes the conversion of wild-type viruses into new recombinant viruses and drives the native viral population to extinction. In this study, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we showed that after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show, however, that targeting evolutionarily conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.

Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism.

The interplay between defense and counterdefense systems of bacteria and bacteriophages has been driving the evolution of both organisms, leading to their great genetic diversity. Restriction-modification systems are well-studied defense mechanisms of bacteria, while phages have evolved covalent modifications as a counterdefense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant, which contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR escape mutations, allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential countermechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counterdefense mechanisms exhibited by bacteria and phages, while affording protection against one another, also provide evolutionary benefits for both. IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well accepted that T4 and other phages have evolved counterdefense mechanisms to protect their genomes from R-M cleavage by covalent modifications, such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRISPR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and it is also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counterdefense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity.

Unlike RNA viruses, most DNA viruses replicate their genomes with high-fidelity polymerases that rarely make base substitution errors. Nevertheless, experimental evolution studies have revealed rapid acquisition of adaptive mutations during serial passage of attenuated vaccinia virus (VACV). One way in which adaptation can occur is by an accordion mechanism in which the gene copy number increases followed by base substitutions and, finally, contraction of the gene copy number. Here, we show rapid acquisition of multiple adaptive mutations mediated by a gene-inactivating frameshift mechanism during passage of an attenuated VACV. Attenuation had been achieved by exchanging the VACV A8R intermediate transcription factor gene with the myxoma virus ortholog. A total of seven mutations in six different genes occurred in three parallel passages of the attenuated virus. The most frequent mutations were single-nucleotide insertions or deletions within runs of five to seven As or Ts, although a deletion of 11 nucleotides also occurred, leading to frameshifts and premature stop codons. During 10 passage rounds, the attenuated VACV was replaced by the mutant viruses. At the end of the experiment, virtually all remaining viruses had one fixed mutation and one or more additional mutations. Although nucleotide substitutions in the transcription apparatus accounted for two low-frequency mutations, frameshifts in genes encoding protein components of the mature virion, namely, A26L, G6R, and A14.5L, achieved 74% to 98% fixation. The adaptive role of the mutations was confirmed by making recombinant VACV with A26L or G6R or both deleted, which increased virus replication levels and decreased particle/PFU ratios. IMPORTANCE Gene inactivation is considered to be an important driver of orthopoxvirus evolution. Whereas cowpox virus contains intact orthologs of genes present in each orthopoxvirus species, numerous genes are inactivated in all other members of the genus. Inactivation of additional genes can occur upon extensive passaging of orthopoxviruses in cell culture leading to attenuation in vivo, a strategy for making vaccines. Whether inactivation of multiple viral genes enhances replication in the host cells or has a neutral effect is unknown in most cases. Using an experimental evolution protocol involving serial passages of an attenuated vaccinia virus, rapid acquisition of inactivating frameshift mutations occurred. After only 10 passage rounds, the starting attenuated vaccinia virus was displaced by viruses with one fixed mutation and one or more additional mutations. The high frequency of multiple inactivating mutations during experimental evolution simulates their acquisition during normal evolution and extensive virus passaging to make vaccine strains.

Hazara nairovirus (HAZV) is a member of the family Nairoviridae in the order Bunyavirales and closely related to Crimean-Congo hemorrhagic fever virus, which is responsible for severe and fatal human disease. The HAZV genome comprises three segments of negative-sense RNA, named S, M, and L, with nontranslated regions (NTRs) flanking a single open reading frame. NTR sequences regulate RNA synthesis and, by analogy with other segmented negative-sense RNA viruses, may direct activities such as virus assembly and innate immune modulation. The terminal-proximal nucleotides of 3′ and 5′ NTRs exhibit extensive terminal complementarity; the first 11 nucleotides are strictly conserved and form promoter element 1 (PE1), with adjacent segment-specific nucleotides forming PE2. To explore the functionality of NTR nucleotides within the context of the nairovirus multiplication cycle, we designed infectious HAZV mutants bearing successive deletions throughout both S segment NTRs. Fitness of rescued viruses was assessed in single-step and multistep growth, which revealed that the 3′ NTR was highly tolerant to change, whereas several deletions of centrally located nucleotides in the 5′ NTR led to significantly reduced growth, indicative of functional disruption. Deletions that encroached upon PE1 and PE2 ablated virus growth and identified additional adjacent nucleotides critical for viability. Mutational analysis of PE2 suggest that its signaling ability relies solely on interterminal base pairing and is an independent cis-acting signaling module. This study represents the first mutagenic analysis of nairoviral NTRs in the context of the infectious cycle, and the mechanistic implications of our findings for nairovirus RNA synthesis are discussed. IMPORTANCE Nairoviruses are a group of RNA viruses that include many serious pathogens of humans and animals, including one of the most serious human pathogens in existence, Crimean-Congo hemorrhagic fever virus. The ability of nairoviruses to multiply and cause disease is controlled in major part by nucleotides that flank the 3′ and 5′ ends of nairoviral genes, called nontranslated regions (NTRs). NTR nucleotides interact with other virus components to perform critical steps of the virus multiplication cycle, such as mRNA transcription and RNA replication, with other roles being likely. To better understand how NTRs work, we performed the first comprehensive investigation of the importance of NTR nucleotides in the context of the entire nairovirus replication cycle. We identified both dispensable and critical NTR nucleotides, as well as highlighting the importance of 3′ and 5′ NTR interactions in virus growth, thus providing the first functional map of the nairovirus NTRs.

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 env gene was done for paired blood and semen samples from antiretroviral therapy (ART)-naive men living in Malawi with (n = 19) and without (n = 5) STI-associated urethritis; for a subset of samples, full-length env genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of proinflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population, indicating a recent bottleneck followed by limited viral replication. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, and where specific genotypes can be amplified, perhaps initially by cellular proliferation but further by limited viral replication. IMPORTANCE HIV-1 infection is a sexually transmitted infection that coexists with other STI. Here, we examined the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.

Letter to the editor. There is no abstract. The summary was showed: SARS-CoV-2 has inevitably mutated during its pandemic spread to cause unpredictable effects on COVID-19 and complicate epidemic control efforts. Here we report that a novel SARS-CoV-2 mutation (ORF3a) appears to be spreading worldwide, which deserves close attention.

The New World mammarenavirus Tacaribe virus (TCRV) has been isolated from fruit bats, mosquitoes, and ticks, whereas all other known New World mammarenaviruses are maintained in rodents. TCRV has not been linked to human disease, but it has been shown to protect against Argentine hemorrhagic fever-like disease in marmosets infected with the New World mammarenavirus Junín virus (JUNV), indicating the potential of TCRV as a live-attenuated vaccine for the treatment of Argentine hemorrhagic fever. Implementation of TCRV as a live-attenuated vaccine or a vaccine vector would be facilitated by the establishment of reverse genetics systems for the genetic manipulation of the TCRV genome. In this study, we developed, for the first time, reverse genetics approaches for the generation of recombinant TCRV (rTCRV). We successfully rescued a wild-type (WT) rTCRV (a trisegmented form of TCRV expressing two reporter genes [r3TCRV]) and a bisegmented TCRV expressing a single reporter gene from a bicistronic viral mRNA (rTCRV/GFP). These reverse genetics approaches represent an excellent tool to investigate the biology of TCRV and to explore its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of other viral infections. Notably, we identified a 39-nucleotide (nt) deletion (Δ39) in the noncoding intergenic region (IGR) of the viral large (L) segment that is required for optimal virus multiplication. Accordingly, an rTCRV containing this 39-nt deletion in the L-IGR (rTCRV/Δ39) exhibited decreased viral fitness in cultured cells, suggesting the feasibility of using this deletion in the L-IGR as an approach to attenuate TCRV, and potentially other mammarenaviruses, for their implementation as live-attenuated vaccines or vaccine vectors. IMPORTANCE To date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever caused by mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin, which is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is nonpathogenic in humans but able to provide protection against Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever, demonstrating the feasibility of using TCRV as a live-attenuated vaccine vector for the treatment of JUNV and potentially other viral infections. Here, we describe for the first time the feasibility of generating recombinant TCRV (rTCRV) using reverse genetics approaches, which paves the way to study the biology of TCRV and also its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of mammarenavirus and/or other viral infections in humans.

Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo. They impact the outcome of natural infections, may help drive virus‐host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification., During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into “infectious” virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification. IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo. They impact the outcome of natural infections, may help drive virus‐host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in protection against HCMV infection and disease. Here we describe a vaccine approach based on the well-characterized modified vaccinia virus Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show that in mice, multiantigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the individual antigens. In addition, we demonstrate that the PC/gB antigen combination of these multiantigenic MVA vectors can enhance the stimulation of humoral immune responses that mediate

The HIV reservoir, which comprises diverse proviruses integrated into the genomes of infected, primarily CD4(+) T cells, is the main barrier to developing an effective HIV cure. Our understanding of the genetics and dynamics of proviruses persisting within distinct CD4(+) T cell subsets, however, remains incomplete. Using single-genome amplification, we characterized subgenomic proviral sequences (nef region) from naive, central memory, transitional memory, and effector memory CD4(+) T cells from five HIV-infected individuals on long-term combination antiretroviral therapy (cART) and compared these to HIV RNA sequences isolated longitudinally from archived plasma collected prior to cART initiation, yielding HIV data sets spanning a median of 19.5 years (range, 10 to 20 years) per participant. We inferred a distribution of within-host phylogenies for each participant, from which we characterized proviral ages, phylogenetic diversity, and genetic compartmentalization between CD4(+) T cell subsets. While three of five participants exhibited some degree of proviral compartmentalization between CD4(+) T cell subsets, combined analyses revealed no evidence that any particular CD4(+) T cell subset harbored the longest persisting, most genetically diverse, and/or most genetically distinctive HIV reservoir. In one participant, diverse proviruses archived within naive T cells were significantly younger than those in memory subsets, while for three other participants we observed no significant differences in proviral ages between subsets. In one participant, “old” proviruses were recovered from all subsets, and included one sequence, estimated to be 21.5 years old, that dominated (>93%) their effector memory subset. HIV eradication strategies will need to overcome within- and between-host genetic complexity of proviral landscapes, possibly via personalized approaches. IMPORTANCE The main barrier to HIV cure is the ability of a genetically diverse pool of proviruses, integrated into the genomes of infected CD4(+) T cells, to persist despite long-term suppressive combination antiretroviral therapy (cART). CD4(+) T cells, however, constitute a heterogeneous population due to their maturation across a developmental continuum, and the genetic “landscapes” of latent proviruses archived within them remains incompletely understood. We applied phylogenetic techniques, largely novel to HIV persistence research, to reconstruct within-host HIV evolutionary history and characterize proviral diversity in CD4(+) T cell subsets in five individuals on long-term cART. Participants varied widely in terms of proviral burden, genetic diversity, and age distribution between CD4(+) T cell subsets, revealing that proviral landscapes can differ between individuals and between infected cell types within an individual. Our findings expose each within-host latent reservoir as unique in its genetic complexity and support personalized strategies for HIV eradication.

Ross River virus (RRV) belongs to the genus Alphavirus and is prevalent in Australia. RRV infection can cause arthritic symptoms in patients and may include rash, fever, arthralgia, and myalgia. Type I interferons (IFN) are the primary antiviral cytokines and trigger activation of the host innate immune system to suppress the replication of invading viruses. Alphaviruses are able to subvert the type I IFN system, but the mechanisms used are ill defined. In this study, seven RRV field strains were analyzed for induction of and sensitivity to type I IFN. The sensitivities of these strains to human IFN-β varied significantly and were highest for the RRV 2548 strain. Compared to prototype laboratory strain RRV-T48, RRV 2548 also induced higher type I IFN levels both in vitro and in vivo and caused milder disease. To identify the determinants involved in type I IFN modulation, the region encoding the nonstructural proteins (nsPs) of RRV 2548 was sequenced, and 42 amino acid differences from RRV-T48 were identified. Using fragment swapping and site-directed mutagenesis, we discovered that substitutions E402A and R522Q in nsP1 as well as Q619R in nsP2 were responsible for increased sensitivity of RRV 2548 to type I IFN. In contrast, substitutions A31T, N219T, S580L, and Q619R in nsP2 led to induction of higher levels of type I IFN. With exception of E402A, all these variations are common for naturally occurring RRV strains. However, they are different from all known determinants of type I IFN modulation reported previously in nsPs of alphaviruses. IMPORTANCE By identifying natural Ross River virus (RRV) amino acid determinants for type I interferon (IFN) modulation, this study gives further insight into the mechanism of type I IFN modulation by alphaviruses. Here, the crucial role of type I IFN in the early stages of RRV disease pathogenesis is further demonstrated. This study also provides a comparison of the roles of different parts of the RRV nonstructural region in type I IFN modulation, highlighting the importance of nonstructural protein 1 (nsP1) and nsP2 in this process. Three substitutions in nsP1 and nsP2 were found to be independently associated with enhanced type I IFN sensitivity, and four independent substitutions in nsP2 were important in elevated type I IFN induction. Such evidence has clear implications for RRV immunobiology, persistence, and pathology. The identification of viral proteins that modulate type I IFN may also have importance for the pathogenesis of other alphaviruses.

Interferon lambda 4 (IFN-λ4) is a recently identified enigmatic member of the interferon (IFN) lambda family. Genetic data suggest that the IFNL4 gene acts in a proviral and anti-inflammatory manner in patients. However, the protein is indistinguishable in vitro from the other members of the interferon lambda family. We have investigated the gene regulation of IFNL4 in detail and found that it differs radically from that of canonical antiviral interferons. Being induced by viral infection is a defining characteristic of interferons, but viral infection or overexpression of members of the interferon regulatory factor (IRF) family of transcription factors only leads to a minute induction of IFNL4. This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the IFNL4 promoter. Thus, the regulation of the IFNL4 gene is radically different and might explain some of the atypical phenotypes associated with the IFNL4 gene in humans. IMPORTANCE Recent genetic evidence has highlighted how the IFNL4 gene acts in a counterintuitive manner, as patients with a nonfunctional IFNL4 gene exhibit increased clearance of hepatitis C virus (HCV) but also increased liver inflammation. This suggests that the IFNL4 gene acts in a proviral and anti-inflammatory manner. These surprising but quite clear genetic data have prompted an extensive examination of the basic characteristics of the IFNL4 gene and its gene product, interferon lambda 4 (IFN-λ4). We have investigated the expression of the IFNL4 gene and found it to be poorly induced by viral infections. A thorough investigation of the IFNL4 promoter revealed a highly conserved and functional promoter, but also one that lacks the defining characteristic of interferons (IFNs), i.e., the ability to be effectively induced by viral infections. We suggest that the unique function of the IFNL4 gene is related to its noncanonical transcriptional regulation.

Type A and type B influenza viruses (FluA and FluB viruses) are two major human pathogens that share common structural and functional features. FluA and FluB viruses can reassort within each type but never between the types. Here, we bioinformatically analyzed all promoter sequences of FluA and FluB viruses and confirmed the presence of the type-specific promoter elements. We then studied the promoter elements with cell-based in vivo assays and an in vitro replication initiation assay. Our results identified, for the first time, a type-specific promoter element—the nucleotide at position 5 in the 3′ end of the viral RNA (vRNA)—that plays a key role(s) in modulating polymerase activity in a type-specific manner. Interestingly, swapping the promoter element between FluA and FluB recombinant viruses showed different tolerances: the replacement of FluA virus-specific U5 with FluB virus-specific C5 in influenza virus A/WSN/33 (H1N1) could be reverted to U5 after 2 to 3 passages, while the replacement of FluB virus-specific C5 with FluA virus-specific U5 in influenza virus B/Yamagata/88 could be maintained, but with significantly reduced replication efficiency. Therefore, our findings indicate that the nucleotide variation at position 5 in the 3′ end of the vRNA promoter between FluA and FluB viruses contributes to their RNP incompatibility, which may shed new light on the mechanisms of intertypic exclusion of reassortment between FluA and FluB viruses. IMPORTANCE Genetic reassortment of influenza virus plays a key role in virus evolution and the emergence of pandemic strains. The reassortment occurs extensively within either FluA or FluB viruses but never between them. Here, we bioinformatically compared available promoter sequences of FluA and FluB viruses and confirmed the presence of the type-specific promoter elements. Our in vivo and in vitro mutagenesis studies showed that a type-specific promoter element—the nucleotide at position 5 in the 3′ end of vRNA promoters—plays key roles in modulating polymerase activity. Interestingly, FluA and FluB viruses showed different tolerances upon key promoter element swapping in the context of virus infections. We concluded that the nucleotide at position 5 in the 3′ end of the vRNA promoters of FluA and FluB viruses is a critical type-specific determinant. This work has implications for further elucidating the mechanisms of the intertypic exclusion of reassortment between FluA and FluB viruses.

The key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies., The polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation. IMPORTANCE The key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

This study demonstrates that the deletion in the PK region occurred naturally in the FMDV genome. The isolated O/ME-SA/PanAsia lineage FMDV with an 86-nt deletion in the PK region showed a pig-adapted characteristic that could cause clinical signs in swine but not bovines. Compared to the wild-type FMDV strain, which possesses full infection capacity in both swine and bovines, the recombinant virus with the 86-nt deletion in the PK region is deficient in causing disease in bovines. Deletion of the previously reported 43 nt in the PK region also led to significantly decreased pathogenicity of FMDV in bovines. This study indicates that the PK region is a novel determinant of the tropism and virulence of FMDV., Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is characterized by genetic instability and different antigenic properties. The nonstructural protein 3A is a primary determinant of the tropism and virulence of Cathay topotype FMDVs. However, several other determinants are also speculated to be involved in viral tropism and virulence. Deletion of 43 nucleotides (nt) in the pseudoknot (PK) region of the 5′ untranslated region (UTR) has been found to coexist with the identified 3A deletion in Cathay topotype FMDV genomes. In this study, we isolated an O/ME-SA/PanAsia lineage FMDV strain, O/GD/CHA/2015, that includes an 86-nt deletion in the PK region and shows a porcinophilic phenotype. To investigate the potential role of the PK region in viral pathogenicity, we generated a recombinant FMDV strain with an incomplete PK region and compared its virulence and pathogenesis to the intact FMDV strain in swine and bovines. Deletion of the 86 nt in the PKs had no major effects on the pathogenicity of the virus in swine but significantly attenuated its ability to infect bovine cells and cattle, indicating that the PK region is a newly discovered determinant of viral tropism and virulence. The role of the 43-nt deletion existing in the Cathay topotype FMDV was also investigated by evaluating the infection properties of genetically engineered viruses. Consistently, the 43-nt deletion in the PK region significantly decreased the pathogenicity of the virus in bovines. Overall, our findings suggest that the PK region deletion occurred naturally in the FMDV genome and that the PK region is highly associated with viral host range and functions as a novel determinant for FMDV pathogenesis. IMPORTANCE This study demonstrates that the deletion in the PK region occurred naturally in the FMDV genome. The isolated O/ME-SA/PanAsia lineage FMDV with an 86-nt deletion in the PK region showed a pig-adapted characteristic that could cause clinical signs in swine but not bovines. Compared to the wild-type FMDV strain, which possesses full infection capacity in both swine and bovines, the recombinant virus with the 86-nt deletion in the PK region is deficient in causing disease in bovines. Deletion of the previously reported 43 nt in the PK region also led to significantly decreased pathogenicity of FMDV in bovines. This study indicates that the PK region is a novel determinant of the tropism and virulence of FMDV.

Epstein-Barr virus is associated with several human malignancies, including nasopharyngeal carcinoma, gastric cancer, and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (oriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half-site. This structure shows that the DNA is smoothly bent, allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised oriP-containing plasmid replication in cells, and impaired recruitment of the MCM3 complex to the oriP. Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the oriP. Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease. IMPORTANCE Epstein-Barr virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of Epstein-Barr virus nuclear antigen 1 (EBNA1) with the viral origin of plasmid replication (oriP). The dyad symmetry (DS) element in oriP is the essential minimal replicator of oriP. Here we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.

Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm, and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results provided not only the mechanism of how mRNA splicing generates viral proteins that have distinct functions but also a novel strategy for replication control of RNA viruses using isoforms with different subcellular localizations. IMPORTANCE Borna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the orthobornavirus genus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than the wild type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small genomes.

Simian virus 40 (SV40) exists as chromatin throughout its life cycle and undergoes typical epigenetic regulation mediated by changes in nucleosome location and associated histone modifications. In order to investigate the role of epigenetic regulation during the encapsidation of late-stage minichromosomes into virions, we mapped the locations of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-h-postinfection minichromosomes and disrupted virions. In minichromosomes obtained late in infection, nucleosomes were found carrying various histone modifications primarily in the regulatory region, with a major nucleosome located within the enhancer and other nucleosomes at the early and late transcriptional start sites. The nucleosome found in the enhancer would be expected to repress early transcription by blocking access to part of the SP1 binding sites and the left side of the enhancer in late-stage minichromosomes while also allowing late transcription. In chromatin from virions, the principal nucleosome located in the enhancer was shifted ∼70 bases in the late direction from what was found in minichromosomes, and the level of modified histones was increased throughout the genome. The shifting of the enhancer-associated nucleosome to the late side would effectively serve as a switch to relieve the repression of early transcription found in late minichromosomes while likely also repressing late transcription by blocking access to necessary regulatory sequences. This epigenetic switch appeared to occur during the final stage of virion formation. IMPORTANCE For a virus to complete infection, it must produce a new virus particle in which the genome is able to support a new infection. This is particularly important for viruses like simian virus 40 (SV40), which exist as chromatin throughout their life cycles, since chromatin structure plays a major role in the regulation of the life cycle. In order to determine the role of SV40 chromatin structure late in infection, we mapped the locations of nucleosomes and their histone tail modifications in SV40 minichromosomes and in the SV40 chromatin found in virions using chromatin immunoprecipitation-DNA sequencing (ChIP-Seq). We have identified a novel viral transcriptional control mechanism in which a nucleosome found in the regulatory region of the SV40 minichromosome is directed to slide during the formation of the virus particle, exposing transcription factor binding sites required for early transcription that were previously blocked by the presence of the nucleosome.

Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide short interfering RNA (siRNA) screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host range restriction factor along with additional candidates, including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knockout (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80S and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained nonpermissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L, or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction. IMPORTANCE A dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the other’s vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA, and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L, or G3 binding proteins, suggesting a novel mechanism of host restriction.

The evolution of mammalian genomes has been shaped by interactions with endogenous retroviruses (ERVs). In this study, we investigated the distribution and diversity of ERVs in the mammalian order Perissodactyla, with a view to understanding their impact on the evolution of modern equids (family Equidae). We characterize the major ERV lineages in the horse genome in terms of their genomic distribution, ancestral genome organization, and time of activity. Our results show that subsequent to their ancestral divergence from rhinoceroses and tapirs, equids acquired four novel ERV lineages. We show that two of these ERV lineages proliferated extensively in the lineage leading to modern horses, and one contains loci that are actively transcribed in specific tissues. In addition, we show that the white rhinoceros has resisted germ line colonization by retroviruses for more than 54 million years—longer than any other extant mammalian species. The map of equine ERVs that we provide here will be of great utility to future studies aiming to investigate the potential functional roles of equine ERVs and their impact on equine evolution. IMPORTANCE ERVs in the host genome are highly informative about the long-term interactions of retroviruses and hosts. They are also interesting because they have influenced the evolution of mammalian genomes in various ways. In this study, we derive a calibrated timeline describing the process through which ERV diversity has been generated in the equine germ line. We determined the distribution and diversity of perissodactyl ERV lineages and inferred their retrotranspositional activity during evolution, thereby gaining insight into the long-term coevolutionary history of retroviruses and mammals. Our study provides a platform for future investigations to identify equine ERV loci involved in physiological processes and/or pathological conditions.

We manipulated SIVmac239Δnef, a model of major histocompatibility complex (MHC)-independent viral control, to evaluate characteristics of effective cellular responses mounted by Mauritian cynomolgus macaques (MCMs) that express the M3 MHC haplotype, which has been associated with poor control of pathogenic simian immunodeficiency virus (SIV). We created SIVΔnef-8x to test the hypothesis that effective SIV-specific T cell responses targeting invariant viral regions can emerge in the absence of immunodominant CD8(+) T cell responses targeting variable epitopes and that control is achievable in individuals lacking known “protective” MHC alleles. Full-proteome gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays identified six newly targeted immunogenic regions following SIVΔnef-8x infection of M3/M3 MCMs. We deep sequenced circulating virus and found that four of the six newly targeted regions rarely accumulated mutations. Six animals infected with SIVΔnef-8x had T cell responses that targeted at least one of the four invariant regions and had a lower set point viral load than two animals that did not have T cell responses that targeted any invariant regions. We found that MHC class II molecules restricted all four of the invariant peptide regions, while the two variable regions were restricted by MHC class I molecules. Therefore, in the absence of immunodominant CD8(+) T cell responses that target variable regions during SIVmac239Δnef infection, individuals without protective MHC alleles developed predominantly CD4(+) T cell responses specific for invariant regions that may improve control of virus replication. Our results provide some evidence that antiviral CD4(+) T cells during acute SIV infection can contribute to effective viral control and should be considered in strategies to combat HIV infection. IMPORTANCE Studies defining effective cellular immune responses to human immunodeficiency virus (HIV) and SIV have largely focused on a rare population that express specific MHC class I alleles and control virus replication in the absence of antiretroviral treatment. This leaves in question whether similar effective immune responses can be achieved in the larger population. The majority of HIV-infected individuals mount CD8(+) T cell responses that target variable viral regions that accumulate high-frequency escape mutations. Limiting T cell responses to these variable regions and targeting invariant viral regions, similar to observations in rare “elite controllers,” may provide an ideal strategy for the development of effective T cell responses in individuals with diverse MHC genetics. Therefore, it is of paramount importance to determine whether T cell responses can be redirected toward invariant viral regions in individuals without protective MHC alleles and if these responses improve control of virus replication.

Japanese macaque (JM) rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from JM with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immunohistopathology comparable to those of multiple sclerosis in humans. Little is known about the molecular biology of JMRV. Here, we sought to identify and characterize the small RNAs expressed during lytic JMRV infection using deep sequencing. Fifteen novel viral microRNAs (miRNAs) were identified in JMRV-infected fibroblasts, all of which were readily detectable by 24 h postinfection and accumulated to high levels by 72 h. Sequence comparisons to human Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs revealed several viral miRNA homologs. To functionally characterize JMRV miRNAs, we screened for their effects on nuclear factor kappa B (NF-κB) signaling in the presence of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Multiple JMRV miRNAs suppressed cytokine-induced NF-κB activation. One of these miRNAs, miR-J8, has seed sequence homology to members of the cellular miR-17/20/106 and miR-373 families, which are key players in cell cycle regulation as well as inflammation. Using reporters, we show that miR-J8 can target 3′ untranslated regions (UTRs) with miR-17-5p or miR-20a cognate sites. Our studies implicate JMRV miRNAs in the suppression of innate antiviral immune responses, which is an emerging feature of many viral miRNAs. IMPORTANCE Gammaherpesviruses are associated with multiple diseases linked to immunosuppression and inflammation, including AIDS-related cancers and autoimmune diseases. JMRV is a recently identified herpesvirus that has been linked to JME, an inflammatory demyelinating disease in Japanese macaques that mimics multiple sclerosis. There are few large-animal models for gammaherpesvirus-associated pathogenesis. Here, we provide the first experimental evidence of JMRV miRNAs in vitro and demonstrate that one of these viral miRNAs can mimic the activity of the cellular miR-17/20/106 family. Our work provides unique insight into the roles of viral miRNAs during rhadinovirus infection and provides an important step toward understanding viral miRNA function in a nonhuman primate model system.

Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCE HIV, like many retroviruses, utilizes a −1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.

The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. IMPORTANCE The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo . Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 are classified as roseoloviruses and are highly prevalent in the human population. Roseolovirus reactivation in an immunocompromised host can cause severe pathologies. While the pathogenic potential of HHV-7 is unclear, it can reactivate HHV-6 from latency and thus contributes to severe pathological conditions associated with HHV-6. Because of the ubiquitous nature of roseoloviruses, their roles in such interactions and the resulting pathological consequences have been difficult to study. Furthermore, the lack of a relevant animal model for HHV-7 infection has hindered a better understanding of its contribution to roseolovirus-associated diseases. Using next-generation sequencing analysis, we characterized the unique genome of an uncultured novel pigtailed macaque roseolovirus. Detailed genomic analysis revealed the presence of gene homologs to all 84 known HHV-7 open reading frames. Phylogenetic analysis confirmed that the virus is a macaque homolog of HHV-7, which we have provisionally named Macaca nemestrina herpesvirus 7 (MneHV7). Using high-throughput RNA sequencing, we observed that the salivary gland tissue samples from nine different macaques had distinct MneHV7 gene expression patterns and that the overall number of viral transcripts correlated with viral loads in parotid gland tissue and saliva. Immunohistochemistry staining confirmed that, like HHV-7, MneHV7 exhibits a natural tropism for salivary gland ductal cells. We also observed staining for MneHV7 in peripheral nerve ganglia present in salivary gland tissues, suggesting that HHV-7 may also have a tropism for the peripheral nervous system. Our data demonstrate that MneHV7-infected macaques represent a relevant animal model that may help clarify the causality between roseolovirus reactivation and diseases. IMPORTANCE Human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 are classified as roseoloviruses. We have recently discovered that pigtailed macaques are naturally infected with viral homologs of HHV-6 and HHV-7, which we provisionally named MneHV6 and MneHV7, respectively. In this study, we confirm that MneHV7 is genetically and biologically similar to its human counterpart, HHV-7. We determined the complete unique MneHV7 genome sequence and provide a comprehensive annotation of all genes. We also characterized viral transcription profiles in salivary glands from naturally infected macaques. We show that broad transcriptional activity across most of the viral genome is associated with high viral loads in infected parotid glands and that late viral protein expression is detected in salivary duct cells and peripheral nerve ganglia. Our study provides new insights into the natural behavior of an extremely prevalent virus and establishes a basis for subsequent investigations of the mechanisms that cause HHV-7 reactivation and associated disease.

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death. IMPORTANCE Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases.