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Narrative structures, though invisible to the naked eye, guide our understanding of pandemics. Like curves and graphs, we can plot them, identify their patterns and organizing principles. These structures act upon our understanding of social and biological events just as much as the rhythms of viral replication and mutation. They order not only themselves but also social and health outcomes. This essay uses narrative precision to expand beyond Charles Rosenberg's influential dramaturgic model and develops new pandemic forms, scaled from the level of an individual line break to the multi-part series: Arc, a form of sequence. Cycle, a form of repetition. Sequel, a form of elongation. Caesura, a form of break. It investigates the potentialities and limitations of these forms, how they intersect, collide, and contradict, and how analysis of these interactions contributes to a deeper understanding of pandemics, their effects, and the diverse perspectives defining their structures. In doing so, it prototypes how literary methods offer conceptual frameworks for pandemic historiography and how a transdisciplinary, medical humanities analysis produces novel understandings at the intersection of health, culture, and society. [ABSTRACT FROM AUTHOR]

As the H9N2 subtype avian influenza virus (H9N2 AIV) evolves naturally, mutations in the hemagglutinin (HA) protein still occur, which involves some sites with glycosylations. It is widely established that glycosylation of the H9N2 AIV HA protein has a major impact on the antigenicity and pathogenicity of the virus. However, the biological implications of a particular glycosylation modification site (GMS) have not been well investigated. In this study, we generated viruses with different GMSs based on wild-type (WT) viruses. Antigenicity studies revealed that the presence of viruses with a 200G+/295G− mutation (with glycosylation at position 200 and deletion of glycosylation at position 295 in the HA protein) combined with a single GMS, such as 87G+, 127G+, 148G+, 178G+, or 265G+, could significantly affect the antigenicity of the virus. Pathogenicity assays revealed that the addition of GMS, such as 127G+, 188G+, 148G+, 178G+, or 54G+, decreased the virulence of the virus in mice, except for 87G+. The removal of GMS, such as 280G− or 295G−, increased the pathogenicity of the virus in mice. Further studies on pathogenicity revealed that 87G+/295G− could also enhance the pathogenicity of the virus. Finally, we selected the WT, WT-87G+, WT-295G−, and WT-87G+/295G− strains as our further research targets to investigate the detailed biological properties of the viruses. GMS, which can enhance viral pathogenicity, did not significantly affect replication or viral stability in vitro but significantly promoted the expression of proinflammatory factors to enhance inflammatory responses in mouse lungs. These findings further deepen our understanding of the influence of the glycosylation of the HA protein of H9N2 AIV on the pathogenicity and antigenicity of the virus in mice. [ABSTRACT FROM AUTHOR]

There is an ongoing debate regarding whether low-level viremia (LLV), in particular persistent LLV, during HIV treatment with optimal adherence originates from low-level viral replication, viral production, or both. We performed an observational study in 30 individuals with LLV who switched to a boosted darunavir (DRV)-based therapy. In-depth virological analyses were used to characterize the viral population and the (activity) of the viral reservoir. Immune activation was examined using cell-bound and soluble markers. The primary outcome was defined as the effect on HIV-RNA and was categorized by responders (<50 cp/mL) or non-responders (>50 cp/mL). At week 24, 53% of the individuals were considered responders, 40% non-responders, and 7% could not be assigned. Sequencing showed no evolution or selection of drug resistance in the non-responders. Production of defective virus with mutations in either the protease (D25N) or RT active site contributed to persistent LLV in two individuals. We show that in about half of the study participants, the switch to a DRV-based regimen resulted in a viral response indicative of ongoing low-level viral replication as the cause of LLV before the switch. Our data confirm that in clinical management, high genetic barrier drugs like DRV are a safe choice, irrespective of the source of LLV. [ABSTRACT FROM AUTHOR]

A study published in the journal MedComm found that a specific mutation in the viral nucleoprotein of avian influenza A viruses (IAVs) allows them to evade the butyrophilin subfamily 3 member A3 (BTN3A3) restriction on virus replication. This mutation, along with other substitutions in the nucleoprotein, enables IAVs to escape from BTN3A3 and potentially increase their zoonotic potential. The research highlights the importance of surveillance for IAV variations associated with BTN3A3 evasion in order to assess the risk of influenza pandemics and protect global public health. The study also identifies other host factors, such as interferon-stimulated genes, that play a role in controlling IAV infection. [Extracted from the article]

Emergence of new variants of COVID-19 is nothing new. Different variants evolved with additional mutations of the virus as part of the viral replication cycle have different infectious properties. JN.1 is the latest COVID-19 variant that seems to circulate the globe contributing to many infections and hospitalizations labeling it a variant of interest by the WHO. The scientific community has yet to understand its full lethal potential if any. In this mini-review, we summarized the facts known so far about this variant and its possible consequences if any for humanity. [ABSTRACT FROM AUTHOR]

SARS-CoV-2, the causative agent of COVID-19 encodes at least 16 nonstructural proteins of variably understood function. Nsp3, the largest nonstructural protein contains several domains, including a SARS-unique domain (SUD), which occurs only in Sarbecovirus. The SUD has a role in preferentially enhancing viral translation. During isolation of mouse-adapted SARS-CoV-2, we isolated an attenuated virus that contained a single mutation in a linker region of nsp3 (nsp3-S676T). The S676T mutation decreased virus replication in cultured cells and primary human cells and in mice. Nsp3-S676T alleviated the SUD translational enhancing ability by decreasing the interaction between two translation factors, Paip1 and PABP1. We also identified a compensatory mutation in the nucleocapsid (N) protein (N-S194L) that restored the virulent phenotype, without directly binding to SUD. Together, these results reveal an aspect of nsp3-N interactions, which impact both SARS-CoV-2 replication and, consequently, pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus that causes highly contagious intestinal infectious disease, involving clinically characterized by diarrhea, dehydration, vomiting, and high mortality to suckling piglets. As a strategy for antiviral therapy, artificial microRNA (amiRNA) mediated suppression of viral replication has recently become increasingly important. In this study, we evaluated the advantages of using an amiRNA vector against PEDV. Methods: In this study, we evaluated the advantages of using an amiRNA vector against PEDV. We designed two single amiRNA sequences for different conserved sequences of the PEDV S and N genes, and tested their inhibitory effects on PEDV in Vero cells. Results: It was obvious from the CCK-8 results that the transient transfection of amiRNA was non-toxic to the cells. In addition, our results showed that the transient expression of two amiRNAs (amiRNA-349 and amiRNA-1447) significantly reduced the expression of viral RNA and protein in the cells. The TCID50 results showed that the release of virus particles into the culture supernatant was significantly reduced, with an effect as high as 90%. To avoid virus mutation escape, the above two single amiRNA sequences were tandem in this study (amiRNA-349 + 1447), enabling a single microRNA to be expressed simultaneously. The real-time PCR and Western blot results showed that the inhibitory effect was significantly enhanced in each of the different time periods. The TCID50 results showed that the release of virus particles in the culture supernatant was significantly reduced at the different time periods. Conclusions: In summary, these results suggest that an RNAi based on amiRNA targeting the conserved region of the virus is an effective method to improve PEDV nucleic acid inhibitors and provide a novel treatment strategy for PEDV infection. [ABSTRACT FROM AUTHOR]

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence–but coming at the cost of optimal replication in humans–and may represent a first step towards a future emergence event. Author summary: Mosquito-borne viruses must replicate in both mosquito and vertebrate hosts to be maintained in nature successfully. When viruses that are typically transmitted by forest dwelling mosquitoes enter urban environments due to deforestation or travel, they must adapt to urban mosquito vectors to transmit effectively. For mosquito-borne viruses, the need to also replicate in a vertebrate host like humans constrains this adaptation process. Towards understanding how the emerging alphavirus, Mayaro virus, might adapt to transmission by the urban mosquito vector, Ae. aegypti, we used natural evolution approaches to identify several viral mutations that impacted replication in both mosquito and vertebrate hosts. We show that a single mutation in the receptor binding domain of E2 increased transmission by Ae. aegypti after bypassing the midgut infection barrier but simultaneously reduced replication and pathology in a mouse model. Mechanistic studies suggested that this mutation decreases the dependence of MAYV on human Mxra8 and the putative MAYV receptor human ApoER2 during replication. This suggests MAYV with this mutation alone is unlikely to be maintained in a natural transmission cycle between mosquitoes and humans. Understanding the adaptive potential of emerging viruses is critical to preventing future pandemics. [ABSTRACT FROM AUTHOR]

Objectives: We evaluated the HIV-1 capsid genetic variability and lenacapavir drug resistance-associated mutations (DRMs) among drug-naive individuals across HIV-1 clades.Methods: A total of 2031 HIV-1 sequences from drug-naive patients were analysed for capsid amino acid modification and the prevalence of lenacapavir DRMs. Amino acid positions with <5% variability were considered as conserved and variability was analysed by HIV-1 clades.Results: Overall, 63% (148/232) of amino acid positions were conserved in the capsid protein. Of note, conservation was consistent in specific binding residues of cellular factors involved in viral replication [CypA (G89, P90), CPSF6 (Q4, N57, N74, A77, K182) and TRIM-NUP153 (R143)], while N183 (12.31%) was the only non-conserved lenacapavir binding residue. The overall prevalence (95% CI) of lenacapavir DRMs was 0.14% (0.05-0.44) (3/2031), with M66I (0.05%) and Q67H (0.05%) observed in subtype C, and T107N (0.05%) observed in CRF01_AE. Moreover, polymorphic mutations M66C (n = 85; 4.18%), Q67K (n = 78; 3.84%), K70R (n = 7; 0.34%), N74R (n = 57; 2.81%) and T107L (n = 82; 4.03%) were observed at lenacapavir resistance-associated positions.Conclusions: The low level of lenacapavir DRMs (<1%) supports its predicted effectiveness for treatment and prevention, regardless of HIV-1 clades. The established conserved regions hence serve as a hallmark for the surveillance of novel mutations potentially relevant for lenacapavir resistance. [ABSTRACT FROM AUTHOR]

The emergence of more transmissible or aggressive variants of SARS‐CoV‐2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS‐CoV‐2. The siRNA can be further modified with receptor ligands such as peptides using CuI‐catalysed click‐chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus‐induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS‐CoV‐2. Furthermore, we show that an ACE2‐binding peptide‐conjugated siRNA is able to reduce virus replication and virus‐induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click‐chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy. [ABSTRACT FROM AUTHOR]

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure. [ABSTRACT FROM AUTHOR]

Integration of the reverse-transcribed genome is a critical step of the retroviral life cycle. Strand-transfer inhibitors (INSTIs) used for antiretroviral therapy inhibit integration but can lead to resistance mutations in the integrase gene, the enzyme involved in this reaction. A significant proportion of INSTI treatment failures, particularly those with second-generation INSTIs, show no mutation in the integrase gene. Here, we show that replication of a selected dolutegravir-resistant virus with mutations in the 39-PPT (polypurine tract) was effective, although no integrated viral DNA was detected, due to the accumulation of unintegrated viral DNA present as 1-LTR circles. Our results show that mutation in the 39-PPT leads to 1-LTR circles and not linear DNA as classically reported. In conclusion, our data provide a molecular basis to explain a new mechanism of resistance to INSTIs, without mutation of the integrase gene and highlights the importance of unintegrated viral DNA in HIV-1 replication. IMPORTANCE Our work highlights the role of HIV-1 unintegrated viral DNA in viral replication. A virus, resistant to strand-transfer inhibitors, has been selected in vitro. This virus highlights a mutation in the 3'PPT region and not in the integrase gene. This mutation modifies the reverse transcription step leading to the accumulation of 1-LTR circles and not the linear DNA. This accumulation of 1-LTR circles leads to viral replication without integration of the viral genome. [ABSTRACT FROM AUTHOR]

During 2015–2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS). Notably, the scale and magnitude of outbreaks in Southeast Asia were significantly smaller compared to those in the Americas. Sequence comparison between epidemic-associated ZIKV strains from Southeast Asia with those from the Americas revealed a methionine to valine substitution at residue position 114 of the NS5 protein (NS5-M114V) in all the American isolates. Using an American isolate of ZIKV (Natal), we investigated the impact of NS5-M114V mutation on virus replication in cells, virulence in interferon (IFN) α/β receptor knockout (Ifnar-/-) mice, as well as replication and transmission potential in Aedes aegypti mosquitoes. We demonstrated that NS5-M114V mutation had insignificant effect on ZIKV replication efficiency in cells, its ability to degrade STAT2, and virulence in vivo, albeit viremia was slightly prolonged in mice. Furthermore, NS5-M114V mutation decreased mosquito infection and dissemination rates and had no effect on virus secretion into the saliva. Taken together, our findings support the notion that NS5-M114V mutation is unlikely to be a major determinant for virus replication and transmission potential. Author summary: Zika virus (ZIKV) emerged to cause outbreaks in Southeast Asia and the Americas during 2015–2016. However, the scale of outbreaks in Southeast Asia were significantly smaller compared to epidemic in the Americas. A methionine to valine amino acid mutation at residue position 114 of the NS5 protein (NS5-M114V) is hypothesized to influence the epidemic outcomes of ZIKV, which led to the large-scale epidemic that occurred in the Americas. By analyzing infection of mammalian and mosquito cells, IFNα/β receptor knockout (Ifnar-/-) mice and Aedes aegypti mosquitoes with engineered ZIKV isolates containing either methionine or valine at residue position 114 of the NS5 protein, we demonstrated that the NS5-M114V mutation did not affect virus replication efficiency and STAT2 degradation in cells, virulence in mice, or virus secretion into the mosquito saliva. The NS5-M114V mutation slightly prolonged viremia in Ifnar-/- mice and reduced mosquito infection rate. Collectively, our findings suggest that the NS5-M114V mutation is unlikely to have significantly influenced the ZIKV epidemic in the Americas. [ABSTRACT FROM AUTHOR]

We documented a hematologic patient with prolonged SARS-CoV-2 viral replication in whom emergence of viral mutations was documented after the consecutive use of antivirals and convalescent plasma. The virus detected in the last of 12 clinical samples (day 237) had accumulated 22 changes in amino acids and 29 in nucleotides. Some of these changes, such as the E484Q, were mutations of concern as defined by WHO. This finding represents an enormous epidemiological threat and poses a major clinical challenge. Combined antiviral strategies, as well as specific strategies related to the diagnostic approach of prolonged infections for this specific population, may be needed. [ABSTRACT FROM AUTHOR]

An Asian Zika virus (ZIKV) isolated from a Thai patient that was serially passaged in Primary Dog Kidney (PDK) cells for attenuation displayed both big and small plaque-forming viruses by the 7th passage. Two small-plaque isolates were selected and purified for characterization as attenuated ZIKV candidates. In vitro growth kinetics showed significantly reduced titers for small-plaque isolates in Vero cells early post-infection compared to the parental ZIKV and a big-plaque isolate, but no significant difference was observed in C6/36 cells. Viral entry experiments elucidate that titer reduction likely occurred due to the diminished entry capabilities of a small-plaque isolate. Additionally, a small-plaque isolate displayed lowered neurovirulence in newborn mice compared to 100% lethality from infection with the parental ZIKV. Genomic analysis revealed the same three unique non-synonymous mutations for both small-plaque isolates: two on the envelope (E) protein at residues 310, alanine to glutamic acid (A310E), and 393, glutamic acid to lysine (E393K), and one on residue 355 of NS3, histidine to tyrosine (H355Y). Three-dimensional (3D) mapping suggests that the E protein mutations located on the receptor-binding and fusion domain III likely affect cell entry, tropism, and virulence. These ZIKV isolates and genotypic markers will be beneficial for vaccine development. [ABSTRACT FROM AUTHOR]

RNA viruses exist as genetically heterogeneous populations due to high mutation rates, and many of these mutations reduce fitness and/or replication speed. However, it is unknown whether mutations can increase replication speed of a virus already well adapted to replication in cultured cells. By sequentially passaging coxsackievirus B3 in cultured cells and collecting the very earliest progeny, we selected for increased replication speed. We found that a single mutation in a viral capsid protein, VP1-F106L, was sufficient for the fast-replication phenotype. Characterization of this mutant revealed quicker genome release during entry compared to wild-type virus, highlighting a previously unappreciated infection barrier. However, this mutation also reduced capsid stability in vitro and reduced replication and pathogenesis in mice. These results reveal a tradeoff between overall replication speed and fitness. Importantly, this approach--selecting for the earliest viral progeny--could be applied to a variety of viral systems and has the potential to reveal unanticipated inefficiencies in viral replication cycles. [ABSTRACT FROM AUTHOR]

The vRNAs of influenza A viruses contain 12 and 13 nucleotide-long sequences at their 3′ and 5′ termini respectively that are highly conserved and constitute the vRNA promoter. These sequences and the next three segment-specific nucleotides show inverted partial complementarity and are followed by several unpaired nucleotides of poorly characterized function at the 3′ end. We have performed systematic point-mutations at the segment-specific nucleotides 15–18 of the 3′-end of a NS-like vRNA segment. All NS-like vRNAs containing mutations at position 15, and some at positions 16–18 showed reduced transcription/replication efficiency in a transfection/infection system. In addition, the replication of recombinant viruses containing mutations at position 15 was impaired both in single and multi-cycle experiments. This reduction was the consequence of a decreased expression of the NS segment. The data indicate that NS1 plays a role in the transcription/replication of its own segment, which elicits a global defect on virus replication. • Influenza A viruses contain segment-specific nucleotides at the 3′ and 5′ ends. • Pairing and unpairing segment-specific nucleotides modulate genome expression. • Mutations at nucleotide 15 of the 3′ end have the highest effect on NS segment expression. • These mutations reduce NS1 expression. • NS1 plays a role in the transcription/replication of its own segment. [ABSTRACT FROM AUTHOR]

Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Importin‐α7 is a component of the nuclear import machinery required for avian‐mammalian adaptation and replicative fitness in human cells. Here, we elucidate the mechanisms by which influenza A viruses may escape replicative restriction in the absence of importin‐α7. To address this question, we assessed viral evolution in mice lacking the importin‐α7 gene. We show that three mutations in particular occur with high frequency in the viral nucleoprotein (NP) protein (G102R, M105K and D375N) in a specific structural area upon in vivo adaptation. Moreover, our findings suggest that the adaptive NP mutations mediate viral escape from importin‐α7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal. However, viral escape from importin‐α7 by mutations in NP is, at least in part, associated with reduced viral replication highlighting the crucial contribution of importin‐α7 to replicative fitness in human cells. [ABSTRACT FROM AUTHOR]

Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation (NS2B-L99V, NS3-S78A, or NS3-D177E). In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle. [ABSTRACT FROM AUTHOR]

Summary: It was repeatedly reported that the hepatitis B virus (HBV) T1719G mutation was very common and related to progression and malignancy of liver disease. However, its effect on viral replication efficiency remains unclear. In this study, we aimed to evaluate the function and mechanisms of the T1719G mutation on viral replication capacity. Wild‐type and T1719G mutation‐bearing HBV1.2× plasmids were transfected into Huh7 and HepG2 cells, respectively, and HBV total RNA, 3.5 kb RNA and supernatant HBV DNA were assessed using real‐time PCR, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured by time‐resolved fluoroimmunoassay. In order to assess Enh II activity and the binding capacity of HNF3β to Enh II sequence, dual‐luciferase assay and Chromatin immunoprecipitation (ChIP)‐PCR were employed, respectively. Simultaneously, the HBx or HBx‐mut (T1719G) plasmid was co‐transfected to evaluate the effect of HBx on viral replication. Our results showed that the T1719G mutation impaired viral replication efficacy compared with the wild type both by reducing Enh II activity and binding capacity of HNF3β with Enh II. And such reduction caused by T1719G mutation could be rescued by HBx protein. Our results show that the T1719G mutation decreases HBV viral replication capacity possibly by mutant HBx protein and altered Enh II activity. [ABSTRACT FROM AUTHOR]

Highlights • We confirmed that phosphorylation modification of the N protein could impair PRRSV growth efficiency in PAMs. • This study is the first to report that phosphorylation modification of the N protein could regulate PRRSV pathogenicity in piglets. • Mutation of PTM sites could be a novel way to attenuate the virus. This strategy will be beneficial to distinguish the vaccine from a wild-type virus. Abstract The porcine respiratory and reproductive syndrome virus (PRRSV) nucleocapsid (N) protein is a multiphosphorylated protein.It has been proved that the phosphorylation of N protein could regulate the growth ability of PRRSV in Marc-145 cells. However, further investigation is needed to determine whether phosphorylation of the N protein could affect PRRSV virulence in piglets. In this study, we confirmed that the mutations could impair PRRSV replication ability in porcine primary macrophages (PAMs) as they did in Marc-145 cells. The animal experiments suggested that the pathogenicity of the mutated virus (A105-120) was significantly reduced compared with parent strain (XH-GD). Our results suggested that the phosphorylation of the N protein contributes to virus replication and virulence. This study is the first to identify a specific modification involved in PRRSV pathogenicity. Mutation of PTMs sites is also a novel way to attenuate PRRSV virulence. The mutations could be a marker in a vaccine. In conclusion, our study will improve our understanding of the molecular mechanisms of PRRSV pathogenicity. [ABSTRACT FROM AUTHOR]

Compared to the replication of double-stranded RNA and DNA viruses, the replication of single-stranded viruses requires the production of a number of intermediate strands that serve as templates for the synthesis of genomic-sense strands. Two theoretical extreme mechanisms for replication for such single-stranded viruses have been proposed; one extreme being represented by the so-called linear stamping machine and the opposite extreme by the exponential growth. Of course, real systems are more complex and examples have been described in which a combination of such extreme mechanisms can also occur: a fraction of the produced progeny resulting from a stamping-machine type of replication that uses the parental genome as template, whereas other fraction of the progeny results from the replication of other progeny genomes. Martínez et al., Sardanyés et al. and Fornés et al. suggested and analyzed a deterministic model of single-stranded RNA (ssRNA) virus intracellular replication that incorporated variability in the replication mechanisms. To explore how stochasticity can affect this mixed-model principal properties, in this paper, we consider the stability of a stochastically perturbed model of ssRNA virus replication within a cell. Using the direct Lyapunov method, we found sufficient conditions for the stability in probability of equilibrium states for this model. This result confirms that this heterogeneous model of single-stranded RNA virus replication is stable with respect to stochastic perturbations of the environment. [ABSTRACT FROM AUTHOR]

Alphavirus infections are characterized by global inhibition of cellular transcription and rapid induction of a cytopathic effect (CPE) in cells of vertebrate origin. Transcriptional shutoff impedes the cellular response to alphavirus replication and prevents establishment of an antiviral state. Chikungunya virus (CHIKV) is a highly pathogenic alphavirus representative, and its nonstructural protein 2 (nsP2) plays critical roles in both inhibition of transcription and CPE development. Previously, we have identified a small peptide in Sindbis virus (SINV) nsP2 (VLoop) that determined the protein's transcriptional inhibition function. It is located in the surface-exposed loop of the carboxy-terminal domain of nsP2 and exhibits high variability between members of different alphavirus serocomplexes. In this study, we found that SINV-specific mutations could not be directly applied to CHIKV. However, by using a new selection approach, we identified a variety of new VLoop variants that made CHIKV and its replicons incapable of inhibiting cellular transcription and dramatically less cytopathic. Importantly, the mutations had no negative effect on RNA and viral replication rates. In contrast to parental CHIKV, the developed VLoop mutants were unable to block induction of type I interferon. Consequently, they were cleared from interferon (IFN)-competent cells without CPE development. Alternatively, in murine cells that have defects in type I IFN production or signaling, the VLoop mutants established persistent, noncytopathic replication. The mutations in nsP2 VLoop may be used for development of new vaccine candidates against alphavirus infections and vectors for expression of heterologous proteins. [ABSTRACT FROM AUTHOR]

Live attenuated vaccines (LAVs), if sufficiently safe, provide the most potent and durable anti-pathogen responses in vaccinees with single immunizations commonly yielding lifelong immunity. Historically, viral LAVs were derived by blind passage of virulent strains in cultured cells resulting in adaptation to culture and a loss of fitness and disease-causing potential in vivo. Mutations associated with these phenomena have been identified but rarely have specific attenuation mechanisms been ascribed, thereby limiting understanding of the attenuating characteristics of the LAV strain and applicability of the attenuation mechanism to other vaccines. Furthermore, the attenuated phenotype is often associated with single nucleotide changes in the viral genome, which can easily revert to the virulent sequence during replication in animals. Here, we have used a rational approach to attenuation of eastern equine encephalitis virus (EEEV), a mosquito-transmitted alphavirus that is among the most acutely human-virulent viruses endemic to North America and has potential for use as an aerosolized bioweapon. Currently, there is no licensed antiviral therapy or vaccine for this virus. Four virulence loci in the EEEV genome were identified and were mutated individually and in combination to abrogate virulence and to resist reversion. The resultant viruses were tested for virulence in mice to examine the degree of attenuation and efficacy was tested by subcutaneous or aerosol challenge with wild type EEEV. Importantly, all viruses containing three or more mutations were avirulent after intracerebral infection of mice, indicating a very high degree of attenuation. All vaccines protected from subcutaneous EEEV challenge while a single vaccine with three mutations provided reproducible, near-complete protection against aerosol challenge. These results suggest that informed mutation of virulence determinants is a productive strategy for production of LAVs even with highly virulent viruses such as EEEV. Furthermore, these results can be directly applied to mutation of analogous virulence loci to create LAVs from other viruses. [ABSTRACT FROM AUTHOR]

Rhinoviruses (RVs) replicate on cytoplasmic membranes derived from the Golgi apparatus. They encode membrane-targeted proteins 2B, 2C, and 3A, which control trafficking and lipid composition of the replication membrane. The virus recruits host factors for replication, such as phosphatidylinositol 4 (PI4)-kinase 3beta (PI4K3b), which boosts PI4-phosphate (PI4P) levels and drives lipid countercurrent exchange of PI4P against cholesterol at endoplasmic reticulum-Golgi membrane contact sites through the lipid shuttling protein oxysterol binding protein 1 (OSBP1). We identified a PI4K3b inhibitor-resistant RV-A16 variant with a single point mutation in the conserved 2B protein near the cytosolic carboxy terminus, isoleucine 92 to threonine (termed 2B[I92T]). The mutation did not confer resistance to cholesterol-sequestering compounds or OSBP1 inhibition, suggesting invariant dependency on the PI4P/cholesterol lipid countercurrents. In the presence of PI4K3b inhibitor, Golgi reorganization and PI4P lipid induction occurred in RV-A16 2B[I92] but not in wild-type infection. The knockout of PI4K3b abolished the replication of both the 2B[I92T] mutant and the wild type. Doxycycline-inducible expression of PI4K3b in PI4K3b knockout cells efficiently rescued the 2B[I92T] mutant and, less effectively, wild-type virus infection. Ectopic expression of 2B[I92T] or 2B was less efficient than that of 3A in recruiting PI4K3b to perinuclear membranes, suggesting a supportive rather than decisive role of 2B in recruiting PI4K3b. The data suggest that 2B tunes the recruitment of PI4K3b to the replication membrane and allows the virus to adapt to cells with low levels of PI4K3b while still maintaining the PI4P/cholesterol countercurrent for establishing Golgi-derived RV replication membranes. [ABSTRACT FROM AUTHOR]

This Medical News feature examines what is known about the efficacy of COVID-19 vaccines against SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

Coxsackievirus B3 (CVB3) is one of the major pathogens of viral myocarditis, lacking specific anti-virus therapeutic options. Increasing evidence has shown an important involvement of the miR-17-92 cluster both in virus infection and cardiovascular development and diseases, while its role in CVB3-induced viral myocarditis remains unclear. In this study, we found that miR-19a and miR-19b were significantly up-regulated in heart tissues of CVB3-infected mice and exerted a significant facilitatory impact on CVB3 biosynthesis and replication, with a more pronounced effect observed in miR-19b, by targeting the encoding region of viral RNA-dependent RNA polymerase 3D (RdRp, 3Dpol) to increase viral genomic RNA stability. The virus-promoting effects were nullified by the synonymous mutations in the viral 3Dpol-encoding region, which corresponded to the seed sequence shared by miR-19a and miR-19b. In parallel, treatment with miR-19b antagomir not only resulted in a noteworthy suppression of CVB3 replication and infection in infected cells, but also demonstrated a significant reduction in the cardiac viral load of CVB3-infected mice, resulting in a considerable alleviation of myocarditis. Collectively, our study showed that CVB3-induced cardiac miR-19a/19b contributed to viral myocarditis via facilitating virus biosynthesis and replication, and targeting miR-19a/19b might represent a novel therapeutic target for CVB3-induced viral myocarditis. • Induced cardiac miR-19a/19b was associated with viral load in CVB3-infected mice. • MiR-19a/19b enhanced CVB3 replication, with miR-19b exhibiting superior efficacy. • MiR-19a/19b increased viral genomic RNA stability by targeting 3Dpol-encoding region. • MiR-19b agomir increased cardiac viral load and aggravated myocarditis in mice. • Anti-miR-19b reduced cardiac viral load and alleviated myocarditis in mice. [ABSTRACT FROM AUTHOR]

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease caused by the SFTS virus (SFTSV). Although fever and thrombocytopenia are the typical manifestations of SFTS, a specific SFTS case with no fever was observed in Zhejiang, China. In this report, we aimed to explore the probable reason for the absence of fever by analyzing the genetic characteristics and temperature sensitivity (ts) of the SFTSV strain ZJ2013-06, which was isolated from the specific case. Phylogenetically, different clusters of SFTSV strains circulated in Zhejiang. ZJ2013-06 was farthest from ZJ2014-02, an isolate belonging to a Chinese dominant cluster, and nearest to the coastal strain NB24/CHN/2013. Ts tests, performed on Vero cells at 37°C and 39°C, indicated that ZJ2013-06 had restricted replication at 39°C. Its viral loads were substantially reduced at 39°C compared with that at 37°C (approximately 100-fold reduction) and were significantly lower than that of ZJ2014-02 at 39°C (P < 0.01). By adaptive culture at 39°C, the induced strain ZJ2013-06-P7 was obtained. Owing to a reverse mutation (S1616), ZJ2013-06-P7 lost the ts of the original strain, displaying similar replication processes with NB24/CHN/2013. The results indicated that the amino acid residue 1616 was related to the ts characteristics of ZJ2013-06. Our study revealed that ZJ2013-06 was temperature-sensitive and may be related to the absence of fever in our case. [ABSTRACT FROM AUTHOR]

The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20Syn and UL24Syn. To test whether UL21 is needed only for the A855V mutant, additional gBSyn derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gBSyn phenotype. This is the first example of a differential requirement for a viral protein across the four syn loci. [ABSTRACT FROM AUTHOR]

The RNA-dependent RNA polymerases of Flaviviridae viruses are crucial for replication. The Flaviviridae polymerase is organized into structural motifs (A-G), with motifs F, A, C and E containing interrogating, priming and catalytic substrate-interacting sites. Modified nucleoside analogues act as antiviral drugs by targeting Flaviviridae polymerases and integrating into the synthesized product causing premature termination. A threonine mutation of a conserved serine residue in motif B of Flaviviridae polymerases renders resistance to 2′- C -methylated nucleoside analogues. The mechanism how this single mutation causes Flaviviridae viruses to escape nucleoside analogues is not yet known. Given the pivotal position of the serine residue in motif B that supports motif F, we hypothesized the threonine mutation causes alterations in nucleoside exploration within the entry tunnel. Implementing a stochastic molecular software showed the all-atom 2′- C -methylated analogue reaction within the active sites of wild type and serine-threonine mutant polymerases from Hepacivirus and Flavivirus. Compared with the wild type, the serine-threonine mutant polymerases caused a significant decrease of analogue contacts with conserved interrogating residues in motif F and a displacement of metal ion cofactors. The simulations significantly showed that during the analogue exploration of the active site the hydrophobic methyl group in the serine-threonine mutant repels water-mediated hydrogen bonds with the 2′- C -methylated analogue, causing a concentration of water-mediated bonds at the substrate-interacting sites. Collectively, the data are an insight into a molecular escape mechanism by Flaviviridae viruses from 2′- C -methylated nucleoside analogue inhibitors. [ABSTRACT FROM AUTHOR]

We consider a spatial model of replication in the RNA World in which polymerase ribozymes use neighbouring strands as templates. Point mutation errors create parasites that have the same replication rate as the polymerase. We have shown previously that spatial clustering allows survival of the polymerases as long as the error rate is below a critical error threshold. Here, we additionally consider errors where a polymerase prematurely terminates replication before reaching the end of the template, creating shorter parasites that are replicated faster than the functional polymerase. In well-known experiments where Qβ RNA is replicated by an RNA polymerase protein, the virus RNA is rapidly replaced by very short non-functional sequences. If the same thing were to occur when the polymerase is a ribozyme, this would mean that termination errors could potentially destroy the RNA World. In this paper, we show that this is not the case in the RNA replication model studied here. When there is continued generation of parasites of all lengths by termination errors, the system can survive up to a finite error threshold, due to the formation of travelling wave patterns; hence termination errors are important, but they do not lead to the inevitable destruction of the RNA World by short parasites. The simplest assumption is that parasite replication rate is inversely proportional to the strand length. In this worst-case scenario, the error threshold for termination errors is much lower than for point mutations. We also consider a more realistic model in which the time for replication of a strand is the sum of a time for binding of the polymerase, and a time for polymerization. When the binding step is considered, termination errors are less serious than in the worst case. In the limit where the binding time is dominant, replication rates are equal for all lengths, and the error threshold for termination is the same as for point mutations. [ABSTRACT FROM AUTHOR]

The article offers information on mutations arise during RNA virus replication along with the viral mutation rates being the source of genetic diversity. Topics discussed include mechanisms for mutation in virus involves mechanisms intrinsic and extrinsic to viral replication; production of less-fit variants; and factors such as cellular chaperones, and recombination could buffer viral populations against deleterious and lethal mutations.

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. [ABSTRACT FROM AUTHOR]

Identifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a high-throughput approach to quantify the selection that monoclonal antibodies exert on all single amino-acid mutations to a viral protein. This approach, mutational antigenic profiling, involves creating all replication-competent protein variants of a virus, selecting with antibody, and using deep sequencing to identify enriched mutations. We use mutational antigenic profiling to comprehensively identify mutations that enable influenza virus to escape four monoclonal antibodies targeting hemagglutinin, and validate key findings with neutralization assays. We find remarkable mutation-level idiosyncrasy in antibody escape: for instance, at a single residue targeted by two antibodies, some mutations escape both antibodies while other mutations escape only one or the other. Because mutational antigenic profiling rapidly maps all mutations selected by an antibody, it is useful for elucidating immune specificities and interpreting the antigenic consequences of viral genetic variation. [ABSTRACT FROM AUTHOR]

In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity. [ABSTRACT FROM AUTHOR]

Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line (HeLa-E8) stably expressing the CDHR3-Y 529 receptor variant, supports propagation of RV-C after infection. C15 clinical or recombinant isolates replicate in HeLa-E8, however progeny yields are lower than those of related strains of RV-A and RV-B. Serial passaging of C15 in HeLa-E8 resulted in stronger cytopathic effects and increased (≥10-fold) virus binding to cells and progeny yields. The adaptation was acquired by two mutations which increased binding (VP1 T 125 K) and replication (3A E 41 K), respectively. A similar 3A mutation engineered into C2 and C41 cDNAs also improved viral replication (2–8 fold) in HeLa but the heparan sulfate mediated cell-binding enhancement by the VP1 change was C15-specific. The findings now enable large-scale cost-effective C15 production by infection and the testing of RV-C infectivity by plaque assay. [ABSTRACT FROM AUTHOR]

Attributes contributing to the current monkeypox virus (MPXV) outbreak remain unknown. It has been established that mutations in viral proteins may alter phenotype and pathogenicity. To assess if mutations in the MPXV DNA replication complex (RC) contribute to the outbreak, we conducted a temporal analysis of available MPXV sequences to identify mutations, generated a DNA replication complex (RC) using structures of related viral and eukaryotic proteins, and structure prediction method AlphaFold. Ten mutations within the RC were identified and mapped onto the RC to infer role of mutations. Two mutations in F8L (RC catalytic subunit), and two in G9R (a processivity factor) were ∼100% prevalent in the 2022 sequences. F8L mutation L108F emerged in 2022, whereas W411L emerged in 2018, and persisted in 2022. L108 is topologically located to enhance DNA binding affinity of F8L. Therefore, mutation L108F can change the fidelity, sensitivity to nucleoside inhibitors, and processivity of F8L. Surface exposed W411L likely affects the binding of regulatory factor(s). G9R mutations S30L and D88 N in G9R emerged in 2022, and may impact the interaction of G9R with E4R (uracil DNA glycosylase). The remaining six mutations that appeared in 2001, reverted to the first (1965 Rotterdam) isolate. Two nucleoside inhibitors brincidofovir and cidofovir have been approved for MPXV treatment. Cidofovir resistance in vaccinia virus is achieved by A314T and A684V mutations. Both A314 and A684 are conserved in MPXV. Therefore, resistance to these drugs in MPXV may arise through similar mechanisms. • L108F in MPXV DNA polymerase emerged in the 2022 outbreak. • L108F can enhance DNA binding affinity, processivity and drug sensitivity of F8L. • Mutation L108F can change the fidelity and sensitivity to nucleoside inhibitors. • G9R mutations that emerged in 2022, are likely to affect interaction of G9R with E4R. [ABSTRACT FROM AUTHOR]

E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a Wimley- White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872GG873 dipeptide, as well as two aromatic residues (871Wand 875W) accessible to solvent. Reverse genetics utilizing a fulllength infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2. [ABSTRACT FROM AUTHOR]

Rearrangements or point mutations in the noncoding control region (NCCR) of BK polyomavirus (BKPyV) have been associated with higher viral loads and more pronounced organ pathology in immunocompromised patients. The respective alterations affect a multitude of transcription factor binding sites (TFBS) but consistently cause increased expression of the early viral gene region (EVGR) at the expense of late viral gene region (LVGR) expression. By mutating TFBS, we identified three phenotypic groups leading to strong, intermediate, or impaired EVGR expression and corresponding BKPyV replication. Unexpectedly, Sp1 TFBS mutants either activated or inhibited EVGR expression when located proximal to the LVGR (sp1-4) or the EVGR (sp1-2), respectively. We now demonstrate that the bidirectional balance of EVGR and LVGR expression is dependent on affinity, strand orientation, and the number of Sp1 sites. Swapping the LVGR-proximal high-affinity SP1-4 with the EVGR-proximal low-affinity SP1-2 in site strand flipping or inserting an additional SP1-2 site caused a rearranged NCCR phenotype of increased EVGR expression and faster BKPyV replication. The 5' rapid amplification of cDNA ends revealed an imperfect symmetry between the EVGR- and LVGR-proximal parts of the NCCR, consisting of TATA and TATA-like elements, initiator elements, and downstream promoter elements. Mutation or deletion of the archetypal LVGR promoter, which is found in activated NCCR variants, abrogated LVGR expression, which could be restored by providing large T antigen (LTag) in trans. Thus, whereas Sp1 sites control the initial EVGR-LVGR expression balance, LTag expression can override inactivation of the LVGR promoter and acts as a key driver of LVGR expression independently of the Sp1 sites and core promoter elements. [ABSTRACT FROM AUTHOR]

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 Gagpolymerase (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. [ABSTRACT FROM AUTHOR]

Like many other viral pathogens, influenza A viruses can form defective interfering particles (DIPs). These particles carry a large internal deletion in at least one of their genome segments. Thus, their replication depends on the co-infection of cells by standard viruses (STVs), which supply the viral protein(s) encoded by the defective segment. However, DIPs also interfere with STV replication at the molecular level and, despite considerable research efforts, the mechanism of this interference remains largely elusive. Here, we present a mechanistic mathematical model for the intracellular replication of DIPs. In this model, we account for the common hypothesis that defective interfering RNAs (DI RNAs) possess a replication advantage over full-length (FL) RNAs due to their reduced length. By this means, the model captures experimental data from yield reduction assays and from studies testing different co-infection timings. In addition, our model predicts that one important aspect of interference is the competition for viral proteins, namely the heterotrimeric viral RNA-dependent RNA polymerase (RdRp) and the viral nucleoprotein (NP), which are needed for encapsidation of naked viral RNA. Moreover, we find that there may be an optimum for both the DI RNA synthesis rate and the time point of successive co-infection of a cell by DIPs and STVs. Comparing simulations for the growth of DIPs with a deletion in different genome segments suggests that DI RNAs derived from segments which encode for the polymerase subunits are more competitive than others. Overall, our model, thus, helps to elucidate the interference mechanism of DI RNAs and provides a novel hypothesis why DI RNAs derived from the polymerase-encoding segments are more abundant in DIP preparations. [ABSTRACT FROM AUTHOR]

Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. [ABSTRACT FROM AUTHOR]

As a result of antiretroviral therapeutic strategies, human immunodeficiency virus type 1 (HIV-1) infection has become a long-term clinically manageable chronic disease for many infected individuals. However, despite this progress in therapeutic control, including undetectable viral loads and CD4+ T-cell counts in the normal range, viral mutations continue to accumulate in the peripheral blood compartment over time, indicating either low level reactivation and/or replication. Using patients from the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort, whom have been sampled longitudinally for more than 7 years, genetic change was modeled against to the dominant integrated proviral quasispecies with respect to selection pressures such as therapeutic interventions, AIDS defining illnesses, and other factors. Phylogenetic methods based on the sequences of the LTR and tat exon 1 of the HIV-1 proviral DNA quasispecies were used to obtain an estimate of an average mutation rate of 5.3 nucleotides (nt)/kilobasepair (kb)/year (yr) prior to initiation of antiretroviral therapy (ART). Following ART the baseline mutation rate was reduced to an average of 1.02 nt/kb/yr. The post-ART baseline rate of genetic change, however, appears to be unique for each patient. These studies represent our initial steps in quantifying rates of genetic change among HIV-1 quasispecies using longitudinally sampled sequences from patients at different stages of disease both before and after initiation of combination ART. Notably, while long-term ART reduced the estimated mutation rates in the vast majority of patients studied, there was still measurable HIV-1 mutation even in patients with no detectable virus by standard quantitative assays. Determining the factors that affect HIV-1 mutation rates in the peripheral blood may lead to elucidation of the mechanisms associated with changes in HIV-1 disease severity. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the "active site loop" (His129 to His144) and "supporting loop" (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs. [ABSTRACT FROM AUTHOR]

The multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1JRCSF (JRCSF) expressing a vif gene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing a vif gene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF with vif mutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSFΔvif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforced vif mutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/ 43D replicated after a prolonged delay with no mutations in vif but instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations in pol viral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA. [ABSTRACT FROM AUTHOR]

Herpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir. [ABSTRACT FROM AUTHOR]

Viral vaccines have traditionally protected against disease, but for viruses that establish latent infection, it is desirable for the vaccine to reduce infection to reduce latent infection and reactivation. While seroconversion has been used in clinical trials of herpes simplex virus (HSV) vaccines to measure protection from infection, this has not been modeled in animal infection systems. To measure the ability of a genital herpes vaccine candidate to protect against various aspects of infection, we established a non-lethal murine model of genital HSV-2 infection, an ELISA assay to measure antibodies specific for infected cell protein 8 (ICP8), and a very sensitive qPCR assay. Using these assays, we observed that immunization with HSV-2 dl 5-29 virus reduced disease, viral shedding, seroconversion, and latent infection by the HSV-2 challenge virus. Therefore, it may be feasible to obtain protection against genital disease, seroconversion and latent infection by immunization, even if sterilizing immunity is not achieved. [ABSTRACT FROM AUTHOR]

Like most other lentiviruses, maedi-visna virus (MVV) requires Vif for replication in natural target cells and in vivo. Here, we show that Vif-deficient MVV accumulates G–A mutations in the sequence context characteristic of ovine APOBEC3, consistent with a role of MVV Vif in neutralizing APOBEC3. We studied two point mutations in the vif gene of MVV. One was a tryptophan to arginine mutation that affects the interaction with APOBEC3 and caused G–A hypermutation. The other mutation was a proline to serine mutation that together with a mutation in the capsid protein caused attenuated replication in fetal ovine synovial (FOS) cells but not in sheep choroid plexus (SCP) cells. There was no hypermutation associated with this mutation. These results suggest that MVV Vif exerts more than one function and that there may be interaction between Vif and the capsid. The results also suggest the involvement of an unknown host factor in MVV Vif function. [ABSTRACT FROM AUTHOR]

Viruses have coevolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field or designed in vitro. In this study, we sought to understand the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability. IMPORTANCE Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. We show that N-linked glycosylation of the bovine leukemia virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing. [ABSTRACT FROM AUTHOR]