Matteo Biolatti

Innate Nuclear Sensor IFI16 Translocates into the Cytoplasm during the Early Stage of In Vitro Human Cytomegalovirus Infection and Is Entrapped in the Egressing Virions during the Late Stage

ABSTRACT Intrinsic immune mechanisms mediated by constitutively expressed proteins termed “restriction factors” provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established. IMPORTANCE Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.

Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability

ABSTRACT A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication. IMPORTANCE The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.

The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1β by Human Cytomegalovirus through Inhibition of NF-kB Activity

Interleukin-1β (IL-1β) is a key effector of the inflammasome complex in response to pathogens and danger signals. Although it is well known that assembly of the inflammasome triggers proteolytic cleavage of the biologically inactive precursor pro-IL-1β into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1β production via the inflammasome is still poorly understood. Here, we show that the infection of human foreskin fibroblasts (HFFs) with a mutant HCMV lacking the tegument protein pp65 (v65Stop) results in higher expression levels of mature IL-1β compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1β. Furthermore, we show that enhanced IL-1β production by the v65Stop mutant is due in part to induction of DNA binding and the transcriptional activity of NF-κB. Lastly, we demonstrate that HCMV infection of HFFs triggers a non-canonical IL-1β activation pathway where caspase-8 promotes IL-1β maturation independently of caspase-1. Altogether, our findings provide novel mechanistic insights into the interplay between HCMV and the inflammasome system and raise the possibility of targeting pp65 to treat HCMV infection.

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.

Modulation of the innate immune response by human cytomegalovirus.

The interplay between human cytomegalovirus (HCMV) and the innate immune response is a critical process that has attracted the attention of many research groups. The emerging scenario is that the immune response of an HCMV-infected host is mediated by a plethora of viral DNA sensors acting as pattern recognition receptors (PRRs), which are capable of inhibiting indirectly viral infection through the activation of two distinct downstream signaling cascades. The first one triggers the production of cytokines, chemokines and interferons (IFNs), while the second one leads to inflammasome complex formation, which in turn promotes the maturation and secretion of pro-inflammatory cytokines such as interleukin-1β (IL-1β). An additional first line of defense against HCMV is represented by a multiplicity of constitutively expressed restriction factors that inhibit viral replication by directly interfering with the activity of essential viral/cellular genes. Here, we take a closer look at some of the most representative intrinsic restriction factors involved in HCMV infection (e.g. IFI16, ND10 complex, viperin and APOBEC3) and review our current understanding of the mechanisms that HCMV has evolved to counteract both IFN and inflammasome responses.