Slow Infection due to Lowering the Amount of Intact versus Empty Particles Is a Characteristic Feature of Coxsackievirus B5 Dictated by the Structural Proteins.

Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from fast-cytolysis-causing echovirus 1 (EV1) with nonstructural proteins from coxsackievirus B5 (CVB5), which shows persistent infection in RD cells. The results showed that the chimera behaved similarly to parental EV1, leading to efficient cytolysis in both permissive A549 and semipermissive RD cells. In contrast to EV1 and the chimera, CVB5 replicated slowly in permissive cells and showed persistent infection in semipermissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 batches constantly contained significant amounts of empty capsids, also in comparison to CVB5's close relative CVB3. During successive passaging of batches containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increase in the amount of empty particles and the lowering of the amount of infective particles are dictated by the CVB5 structural proteins, leading to slowing down of the infection between passages. Furthermore, the key factor for persistent infection is the small amount of infective particles produced, not the high number of empty particles that accumulate.IMPORTANCE Enteroviruses cause several severe diseases, with lytic infections that lead to rapid cell death but also persistent infections that are more silent and lead to chronic states of infection. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera with the structural proteins of echovirus 1 and the nonstructural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to the production of a high number of empty viruses (empty capsids) that do not contain genetic material and are unable to continue the infection. Coinciding with the high number of empty capsids, the amount of infective virions decreased. This characteristic property was not observed in the constructed chimera virus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infections is essential in understanding virus-caused pathologies.

A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits.

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes.IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.

Strategy of Human Cytomegalovirus To Escape Interferon Beta-Induced APOBEC3G Editing Activity.

The apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) gene products and, to a lesser degree, those of A3F but not of A3A, are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon beta (IFN-ß), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-ß by limiting the presence of A3G hot spots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections.IMPORTANCE APOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study, we show that particularly the APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and that its upregulation is mediated by IFN-ß. Furthermore, we were able to demonstrate that neither A3G knockout nor A3G overexpression appears to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hot spots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteracting mechanisms under A3G-mediated selective pressure.

Enterovirus D68 capsid formation and stability requires acidic compartments.

Enterovirus D68 (EV-D68), a picornavirus traditionally associated with respiratory infections, has recently been linked to a polio-like paralytic condition known as acute flaccid myelitis (AFM). EV-D68 is understudied, and much of the field's understanding of this virus is based on studies of poliovirus. For poliovirus, we previously showed that low pH promotes virus capsid maturation, but here we show that, for EV-D68, inhibition of compartment acidification during a specific window of infection causes a defect in capsid formation and maintenance. These phenotypes are accompanied by radical changes in the infected cell, with viral replication organelles clustering in a tight juxtanuclear grouping. Organelle acidification is critical during a narrow window from 3-4hpi, which we have termed the "transition point," separating translation and peak RNA replication from capsid formation, maturation and egress. Our findings highlight that acidification is crucial only when vesicles convert from RNA factories to virion crucibles.

Starvation after infection restricts enterovirus D68 replication.

Enterovirus D68 (EV-D68) is a respiratory pathogen associated with acute flaccid myelitis, a childhood paralysis disease. No approved vaccine or antiviral treatment exists against EV-D68. Infection with this virus induces the formation of autophagosomes to enhance its replication but blocks the downstream autophagosome- lysosome fusion steps. Here, we examined the impact of autophagy induction through starvation, either before (starvation before infection, SBI) or after (starvation after infection, SAI) EV-D68 infection. We showed that SAI, but not SBI, attenuated EV-D68 replication in multiple cell lines and abrogated the viral-mediated cleavage of host autophagic flux-related proteins. Furthermore, SAI induced autophagic flux during EV-D68 replication and prevented production of virus-induced membranes, which are required for picornavirus replication. Pharmacological inhibition of autophagic flux during SAI did not rescue EV-D68 titers. SAI had the same effect in multiple cell types, and restricted the replication of several medically relevant picornaviruses. Our results highlight the significance of autophagosomes for picornavirus replication and identify SAI as an attractive broad-spectrum anti-picornavirus strategy.Abbreviations: BAF: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CQ: chloroquine; CVB3: coxsackievirus B3; EV-D68: enterovirus D68; hpi: hour post-infection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; NSP2B: nonstructural protein 2B; PV: poliovirus; RES: resveratrol; RV14: rhinovirus 14; SAI: starvation after infection; SBI: starvation before infection; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB.

Enterovirus D68 capsid formation and stability requires acidic compartments.

IMPORTANCE: The respiratory picornavirus enterovirus D68 is a causative agent of acute flaccid myelitis, a childhood paralysis disease identified in the last decade. Poliovirus, another picornavirus associated with paralytic disease, is a fecal-oral virus that survives acidic environments when passing from host to host. Here, we follow up on our previous work showing a requirement for acidic intracellular compartments for maturation cleavage of poliovirus particles. Enterovirus D68 requires acidic vesicles for an earlier step, assembly, and maintenance of viral particles themselves. These data have strong implications for the use of acidification blocking treatments to combat enterovirus diseases.

SIRT-1 is required for release of enveloped enteroviruses.

Enterovirus D68 (EV-D68) is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1's proviral activity is, we demonstrate, mediated through the repression of endoplasmic reticulum stress (ER stress). Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.

Replication properties and immunomodulatory effects of human cytomegalovirus infection impact the clinical presentation in congenital patients: A case series.

BACKGROUND: Human cytomegalovirus (HCMV) is the leading cause of congenital infections resulting in severe morbidity and mortality among newborns worldwide. Although both the host's and the virus' genetic backgrounds contribute to the outcome of infections, significant gaps remain in our understanding of the exact mechanisms that determine disease severity. OBJECTIVES: In this study, we sought to identify a correlation between the virological features of different HCMV strains with the clinical and pathological features of congenitally infected newborns, therefore proposing new possible prognostic factors. STUDY DESIGN: This short communication presents five newborns with congenital cytomegalovirus infection, whose clinical phenotype during fetal, neonatal, and follow-up periods is correlated with in-vitro growth properties, immunomodulatory abilities and genome variability of HCMV strains isolated from organic samples (urine) of the patients. RESULTS: The five patients described in this short communication displayed a heterogeneous clinical phenotype and different virus replication properties, immunomodulatory abilities, and genetic polymorphisms. Interestingly, we observed that an attenuate viral replication in-vitro influences the immunomodulatory abilities of HCMV, leading to more severe congenital infections and long-term sequelae. Conversely, infection with viruses characterized by aggressive replicative behavior in-vitro resulted in asymptomatic patients' phenotypes. CONCLUSIONS: Overall, this case series suggests the hypothesis that genetic variability and differences in the replicative behavior of HCMV strains result in clinical phenotypes of different severity, most likely due to different immunomodulatory properties of the virus.

Genetic Variability of Human Cytomegalovirus Clinical Isolates Correlates With Altered Expression of Natural Killer Cell-Activating Ligands and IFN-γ.

Human cytomegalovirus (HCMV) infection often leads to systemic disease in immunodeficient patients and congenitally infected children. Despite its clinical significance, the exact mechanisms contributing to HCMV pathogenesis and clinical outcomes have yet to be determined. One of such mechanisms involves HCMV-mediated NK cell immune response, which favors viral immune evasion by hindering NK cell-mediated cytolysis. This process appears to be dependent on the extent of HCMV genetic variation as high levels of variability in viral genes involved in immune escape have an impact on viral pathogenesis. However, the link between viral genome variations and their functional effects has so far remained elusive. Thus, here we sought to determine whether inter-host genetic variability of HCMV influences its ability to modulate NK cell responses to infection. For this purpose, five HCMV clinical isolates from a previously characterized cohort of pediatric patients with confirmed HCMV congenital infection were evaluated by next-generation sequencing (NGS) for genetic polymorphisms, phylogenetic relationships, and multiple-strain infection. We report variable levels of genetic characteristics among the selected clinical strains, with moderate variations in genome regions associated with modulation of NK cell functions. Remarkably, we show that different HCMV clinical strains differentially modulate the expression of several ligands for the NK cell-activating receptors NKG2D, DNAM-1/CD226, and NKp30. Specifically, the DNAM-1/CD226 ligand PVR/CD155 appears to be predominantly upregulated by fast-replicating ("aggressive") HCMV isolates. On the other hand, the NGK2D ligands ULBP2/5/6 are downregulated regardless of the strain used, while other NK cell ligands (i.e., MICA, MICB, ULBP3, Nectin-2/CD112, and B7-H6) are not significantly modulated. Furthermore, we show that IFN-γ; production by NK cells co-cultured with HCMV-infected fibroblasts is directly proportional to the aggressiveness of the HCMV clinical isolates employed. Interestingly, loss of NK cell-modulating genes directed against NK cell ligands appears to be a common feature among the "aggressive" HCMV strains, which also share several gene variants across their genomes. Overall, even though further studies based on a higher number of patients would offer a more definitive scenario, our findings provide novel mechanistic insights into the impact of HCMV genetic variability on NK cell-mediated immune responses.

Where do we Stand after Decades of Studying Human Cytomegalovirus?

Human cytomegalovirus (HCMV), a linear double-stranded DNA betaherpesvirus belonging to the family of Herpesviridae, is characterized by widespread seroprevalence, ranging between 56% and 94%, strictly dependent on the socioeconomic background of the country being considered. Typically, HCMV causes asymptomatic infection in the immunocompetent population, while in immunocompromised individuals or when transmitted vertically from the mother to the fetus it leads to systemic disease with severe complications and high mortality rate. Following primary infection, HCMV establishes a state of latency primarily in myeloid cells, from which it can be reactivated by various inflammatory stimuli. Several studies have shown that HCMV, despite being a DNA virus, is highly prone to genetic variability that strongly influences its replication and dissemination rates as well as cellular tropism. In this scenario, the few currently available drugs for the treatment of HCMV infections are characterized by high toxicity, poor oral bioavailability, and emerging resistance. Here, we review past and current literature that has greatly advanced our understanding of the biology and genetics of HCMV, stressing the urgent need for innovative and safe anti-HCMV therapies and effective vaccines to treat and prevent HCMV infections, particularly in vulnerable populations.

Risk of Symptomatic Infection after Non-Primary Congenital Cytomegalovirus Infection.

Cytomegalovirus (CMV) is the leading cause of congenital infection. Its occurrence is phenotypically heterogeneous. The type of maternal infection, primary or non-primary, is an important factor related to the symptomatic disease, the primary infection was long considered the only cause of severe neonatal disease. We aimed to analyze the association of primary and non-primary infection with pathological outcomes in infants and with long-term sequelae at follow-up. This was a monocentric retrospective observational study on a population of 91 infants diagnosed with a CMV infection at the Neonatal Care Unit of Neonatology at the Sant'Anna Hospital of Turin during the period of June 2005 to December 2018. Infants underwent clinical, laboratory, and neuroradiological evaluations at birth. Subsequently, the patients were monitored in an auxological, neurodevelopment, and audiological follow-up. Regarding primary vs. non-primary infection, we found a higher percentage of incidence of symptomatic and neurological localized infection, as well as long-term sequelae in the latter. However, no significant difference between the two populations was found. We underline the possibility of re-infection in previously immunized mothers (non-primary infection) with unfavorable neonatal and long-term outcomes.

Tuning the Orchestra: HCMV vs. Innate Immunity.

Understanding how the innate immune system keeps human cytomegalovirus (HCMV) in check has recently become a critical issue in light of the global clinical burden of HCMV infection in newborns and immunodeficient patients. Innate immunity constitutes the first line of host defense against HCMV as it involves a complex array of cooperating effectors - e.g., inflammatory cytokines, type I interferon (IFN-I), natural killer (NK) cells, professional antigen-presenting cells (APCs) and phagocytes - all capable of disrupting HCMV replication. These factors are known to trigger a highly efficient adaptive immune response, where cellular restriction factors (RFs) play a major gatekeeping role. Unlike other innate immunity components, RFs are constitutively expressed in many cell types, ready to act before pathogen exposure. Nonetheless, the existence of a positive regulatory feedback loop between RFs and IFNs is clear evidence of an intimate cooperation between intrinsic and innate immunity. In the course of virus-host coevolution, HCMV has, however, learned how to manipulate the functions of multiple cellular players of the host innate immune response to achieve latency and persistence. Thus, HCMV acts like an orchestra conductor able to piece together and rearrange parts of a musical score (i.e., innate immunity) to obtain the best live performance (i.e., viral fitness). It is therefore unquestionable that innovative therapeutic solutions able to prevent HCMV immune evasion in congenitally infected infants and immunocompromised individuals are urgently needed. Here, we provide an up-to-date review of the mechanisms regulating the interplay between HCMV and innate immunity, focusing on the various strategies of immune escape evolved by this virus to gain a fitness advantage.

Biological relevance of Cytomegalovirus genetic variability in congenitally and postnatally infected children.

BACKGROUND: Human cytomegalovirus (HCMV) is the leading cause of congenital infections resulting in severe morbidity and mortality among infected children. Although the virus is highly polymorphic, particularly in genes contributing to immune evasion, the mechanisms underlying its genetic variability and pathogenicity are only partially understood. OBJECTIVES: We aimed to characterize different HCMV clinical strains isolated from 21 congenitally- or postnatally-infected children for in vitro growth properties and genetic polymorphisms. STUDY DESIGN: The growth of various HCMV isolates was analyzed in different cell culture models. Genetic polymorphism was assessed by genetic and phylogenetic analysis of viral genes involved in virulence (UL144, US28, and UL18), latency (UL133-138), or drug resistance (UL54 and UL97). RESULTS: Here, we report a high degree of genetic and phenotypic diversity in distinct HCMV clinical isolates, as shown by their in vitro growth properties. In particular, HCMV isolates displayed the highest degree of genetic variability in the UL144 gene, where we were able to define four distinct genotypes within the cohort based on UL144 heterogeneity. Lastly, among all isolates we were able to identify 36 mutations in UL54 and 2 in UL97. CONCLUSIONS: Our findings indicate that surprisingly high levels of genetic HCMV variability correlate with a high degree of phenotypic polymorphism, which in turn might differentially influence the growth, fitness, and drug susceptibility of HCMV.