Integrin Activation and Viral Infection

Integrins are members of a ubiquitous membrane receptor family which includes 18 different α subunits and 8 β subunits forming more than 20 α/β heterodimers. Integrins play key functions in vascular endothelial cell and tumour cell adhesion, lymphocyte trafficking, tumor growth and viral infection. Current understanding of the molecular basis of integrins as viral receptors has been achieved through many decades of study into the biology of transmembrane glycoproteins and their interactions with several viruses. This review provides a summary of the current knowledge on the molecular bases of interactions between viruses and integrins, which are of potential practical significance. Inhibition of virus-integrin interactions at the points of virus attachment or entry will provide a novel approach for the therapeutic treatment of viral diseases.     

Papillomavirus Infection Requires γ Secretase

The mechanism by which papillomaviruses breach cellular membranes to deliver their genomic cargo to the nucleus is poorly understood. Here, we show that infection by a broad range of papillomavirus types requires the intramembrane protease γ secretase. The γ-secretase inhibitor (S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide (compound XXI) inhibits infection in vitro by all types of papillomavirus pseudovirions tested, with a 50% inhibitory concentration (IC₅₀) of 130 to 1,000 pM, regardless of reporter construct and without impacting cellular viability. Conversely, XXI does not inhibit in vitro infection by adenovirus or pseudovirions derived from the BK or Merkel cell polyomaviruses. Vaginal application of XXI prevents infection of the mouse genital tract by human papillomavirus type 16 (HPV16) pseudovirions. Nicastrin and presenilin-1 are essential components of the γ-secretase complex, and mouse embryo fibroblasts deficient in any one of these components were not infected by HPV16, whereas wild-type and β-secretase (BACE1)-deficient cells were susceptible. Neither the uptake of HPV16 into Lamp-1-positive perinuclear vesicles nor the disassembly of capsid to reveal both internal L1 and L2 epitopes and bromodeoxyuridine (BrdU)-labeled encapsidated DNA is dependent upon γ-secretase activity. However, blockade of γ-secretase activity by XXI prevents the BrdU-labeled DNA encapsidated by HPV16 from reaching the ND10 subnuclear domains. Since prior studies indicate that L2 is critical for endosomal escape and targeting of the viral DNA to ND10 and that γ secretase is located in endosomal membranes, our findings suggest that either L2 or an intracellular receptor are cleaved by γ secretase as papillomavirus escapes the endosome.The necessary causal association of persistent infection by an “oncogenic” type of human papillomavirus (HPV) with cervical cancer is firmly established (52, 53). HPV is the most prevalent sexually transmitted infection, and although the majority of patients clear their infection, HPV is directly responsible for 5% of all cancer deaths worldwide (30). HPV is also associated with multiple other anogenital cancers and oropharyngeal cancers.The life cycle of HPV is closely linked to epithelial differentiation within stratified squamous epithelia (16). Initial infection occurs within the undifferentiated proliferative basal cell layer in which only the viral early proteins are expressed, whereas production of the late proteins and, thus, progeny virus is restricted to the terminally differentiated suprabasal compartment (53). The exquisite dependence of virion production upon epithelial differentiation and lack of a rapid phenotype in culture can be circumvented by ectopic expression of the capsid proteins L1 and L2 in cells maintaining viral genome or reporter constructs as episomes, resulting in “quasivirions” or “pseudovirions,” respectively, whose infectivity can be readily and rapidly quantified in vitro or in vivo (6, 11, 35, 41).The completion of the entire papillomavirus life cycle is species specific. However, studies with bovine papillomavirus (BPV) in horses and hamsters, HPV pseudovirions in mouse challenge models, and quasivirions in rabbits suggest that virion internalization and delivery of the encapsidated DNA to the nucleus are promiscuous and that tropism is determined at a later stage of the life cycle (11, 27, 29, 39).Although significant progress has been made in understanding the HPV life cycle and virion structure, many of the molecular events of virus internalization and infection are poorly defined (43). Both the L1 (major) and L2 (minor) capsid proteins provide essential functions during infection (41) (8). L1 is sufficient to form empty capsids, termed virus-like particles (VLPs) (25), which bind to basement membrane and to the cell surface and which also form the basis of the licensed HPV vaccines (10). Glycosaminoglycans (GAGs), most notably heparan sulfate (HS), play a critical role in virion binding and infection, both in vitro and in the murine vaginal challenge model, although differences between HPV types and target cells in vitro have been described (14, 19, 20), for example, between HPV16 and HPV31 (4, 34, 42). Once bound to the basement membrane, the virions undergo a conformation change resulting in the surface display of the amino terminus of L2 and its cleavage by a proprotein convertase (PC), furin and/or PC5/PC6, and the transfer of virions to the cell surface (24). The uptake of the virions is apparently slow as late addition of neutralizing antibodies several hours after initial cell surface binding prevents infection in vitro (9). The endocytic mechanisms reported for various papillomavirus types are diverse, but furin cleavage of L2 and endosomal acidification are critical shared steps (15, 38). In a late endosomal compartment, the L1 capsid disassembles, releasing L2 associated with the previously encapsidated DNA to gain access to the nucleus by an unknown mechanism and to accumulate at the subnuclear domain, ND10 (13). Although L2 contains a C-terminal nuclear localization signal (17), entry to mitosis, which is associated with the dissolution of the nuclear membrane, is required for infection, suggesting that the complex with the viral nucleohistone core is unable pass through nuclear pores (36). It is unclear how the L2-genome complex escapes the endocytic compartment, but the carboxy terminus of L2 also contains both DNA binding and a membrane-destabilizing peptide (21).γ Secretase is an intramembranously cleaving protease (I-CliP) linked to Alzheimer''s disease through its cleavage of amyloid precursor protein (APP) (1). It is a multicomponent complex, and presenilin (PS) is the catalytic unit whose active site contains two aspartate residues. In addition to the nine-pass transmembrane protein PS, γ secretase requires nicastrin (NCT), anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 in an equimolar ratio for proteolytic activity (28). The subcellular localization of γ secretase is controversial but includes the endoplasmic reticulum (23), endosome (26), lysosome (31), and plasma membrane (37), all of which are subcellular locales possibly traversed by papillomavirus during infection (43).By analogy to the cleavage of L2 by furin that is critical for exit from the endosomes (38), we hypothesized that I-CLiP might contribute to papillomavirus infection. Here, we report that a γ-secretase inhibitor prevents HPV infection both in vitro and in the mouse vaginal challenge model and that cell lines lacking essential components of γ secretase are refractory to HPV infection.     

Trypanosoma cruzi Infection through the Oral Route Promotes a Severe Infection in Mice: New Disease Form from an Old Infection?

Oral transmission of Chagas disease has been documented in Latin American countries. Nevertheless, significant studies on the pathophysiology of this form of infection are largely lacking. The few studies investigating oral route infection disregard that inoculation in the oral cavity (Oral infection, OI) or by gavage (Gastrointestinal infection, GI) represent different infection routes, yet both show clear-cut parasitemia and heart parasitism during the acute infection. Herein, BALB/c mice were subjected to acute OI or GI infection using 5x10⁴ culture-derived Trypanosoma cruzi trypomastigotes. OI mice displayed higher parasitemia and mortality rates than their GI counterparts. Heart histopathology showed larger areas of infiltration in the GI mice, whereas liver lesions were more severe in the OI animals, accompanied by higher Alanine Transaminase and Aspartate Transaminase serum contents. A differential cytokine pattern was also observed because OI mice presented higher pro-inflammatory cytokine (IFN-γ, TNF) serum levels than GI animals. Real-time PCR confirmed a higher TNF, IFN-γ, as well as IL-10 expression in the cardiac tissue from the OI group compared with GI. Conversely, TGF-β and IL-17 serum levels were greater in the GI animals. Immunolabeling revealed macrophages as the main tissue source of TNF in infected mice. The high mortality rate observed in the OI mice paralleled the TNF serum rise, with its inhibition by an anti-TNF treatment. Moreover, differences in susceptibility between GI versus OI mice were more clearly related to the host response than to the effect of gastric pH on parasites, since infection in magnesium hydroxide-treated mice showed similar results. Overall, the present study provides conclusive evidence that the initial site of parasite entrance critically affects host immune response and disease outcome. In light of the occurrence of oral Chagas disease outbreaks, our results raise important implications in terms of the current view of the natural disease course and host-parasite relationship.     

Did Gause Have a Yeast Infection?

We planned to develop predator–prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator–prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia‐yeast time‐series data, from Gause. We hypothesised that if the model simulated predator–prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self‐sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator–prey dynamics.     

Measurement of γHV68 Infection in Mice

γ-Herpesviruses (γ-HVs) are notable for their ability to establish latent infections of lymphoid cells¹. The narrow host range of human γ-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine γ-herpesvirus 68 (γHV68) shares extensive genetic and biological similarities with human γ-HVs and is a natural pathogen of murid rodents². As such, evaluation of γHV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during γ-HVs infection.Upon intranasal inoculation, γHV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host^3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of γHV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.Download video file.^{(81M, mov)}     

Herpes Simplex Virus-1 Fine-Tunes Host’s Autophagic Response to Infection: A Comprehensive Analysis in Productive Infection Models

Herpes simplex virus-1 (HSV-1) infection causes severe conditions, with serious complications, including corneal blindness from uncontrolled ocular infections. An important cellular defense mechanism against HSV-1 infection is autophagy. The autophagic response of the host cell was suggested to be regulated by HSV-1. In this study, we performed a detailed analysis of autophagy in multiple HSV-1-targeted cell types, and under various infection conditions that recapitulate a productive infection model. We found that autophagy was slightly inhibited in one cell type, while in other cell types autophagy maintained its basal levels mostly unchanged during productive infection. This study refines the concept of HSV-1-mediated autophagy regulation to imply either inhibition, or prevention of activation, of the innate immune pathway.     

Process of Infection with Bacteriophage φX174: XXXVI. Measurement of Virus-Specific Proteins During a Normal Cycle of Infection

Double-labeling techniques in which (14)C-labeled, phiX174-infected cells and (3)H-labeled, uninfected cells were used permitted the identification of the virus-specific proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without prior inhibition of host-cell protein synthesis by ultraviolet irradiation. It was also possible to detect previously undescribed components of high molecular weight which may represent induced host proteins. The gel regions specifically corresponding to cistron II protein and the chloramphenicol-resistant VI protein were identified, and a third new, small peak of unknown origin was detected. Studies of the rate of synthesis of virus-specific proteins at various times after infection indicated that the product of cistron I (lysis) is made only late in infection, but the other proteins seemed to be synthesized at the same relative rates throughout infection (although in different amounts). Studies of the proteins obtained from uniformly labeled phiX virus particles indicated that all of the spikes are identical and allowed a formulation of the structure of the phage capsid.     

Control of Murine Cytomegalovirus Infection by γδ T Cells

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1^-/- mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1^-/- mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.     

Process of Infection with Bacteriophage φX174: XXXIV. Kinetics of the Attachment and Eclipse Steps of the Infection

The products of phiX cistrons II, III, and VII are demonstrated to affect the attachment of the phage to its host Escherichia coli C; therefore, by inference, these cistrons influence, directly or indirectly, the structure of proteins in the virus particle. Two of the mutations which alter attachment kinetics, ts79 in cistron III and h in cistron VII, also affect the electrophoretic mobility of the virus and emphasize the role of charge in the attachment interaction with the host. The kinetics for attached phage to go into "eclipse" are first-order and biphasic; about 85% of the phage eclipse at one rate (k(e) = 0.86 min(-1)) and the remainder do so at a distinctly lower rate (k(e) = 0.21 min(-1)). No phiX cistrons yet identified affect the eclipse process. The lowest temperature at which eclipse is detected is 19 C. The Arrhenius activation energy for phage eclipse has the high value of 36.6 kcal/mole, indicating the cooperative nature of the event.     

Probucol-Induced α-Tocopherol Deficiency Protects Mice against Malaria Infection

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.     

Type I Interferon Induction during Influenza Virus Infection Increases Susceptibility to Secondary Streptococcus pneumoniae Infection by Negative Regulation of γδ T Cells

The majority of deaths following influenza virus infection result from secondary bacterial superinfection, most commonly caused by Streptococcus pneumoniae. Several models have been proposed to explain how primary respiratory viral infections exacerbate secondary bacterial disease, but the mechanistic explanations have been contradictory. In this study, mice were infected with S. pneumoniae at different days after primary influenza A (X31) virus infection. Our findings show that the induction of type I interferons (IFNs) during a primary nonlethal influenza virus infection is sufficient to promote a deadly S. pneumoniae secondary infection. Moreover, mice deficient in type I interferon receptor (IFNAR knockout [KO] mice) effectively cleared the secondary bacterial infection from their lungs, increased the recruitment of neutrophils, and demonstrated an enhanced innate expression of interleukin-17 (IL-17) relative to wild-type (WT) mice. Lung γδ T cells were responsible for almost all IL-17 production, and their function is compromised during secondary S. pneumoniae infection of WT but not IFNAR KO mice. Adoptive transfer of γδ T cells from IFNAR KO mice reduced the susceptibility to secondary S. pneumoniae infection in the lung of WT mice. Altogether, our study highlights the importance of type I interferon as a key master regulator that is exploited by opportunistic pathogens such as S. pneumoniae. Our findings may be utilized to design effective preventive and therapeutic strategies that may be beneficial for coinfected patients during influenza epidemics.     

What Triggers a Diagnosis of HIV Infection in the Tokyo Metropolitan Area? Implications for Preventing the Spread of HIV Infection in Japan

Background

Japan has not succeeded in reducing the annual number of new HIV-infected patients, although the prevalence of HIV infection is low (0.02%).

Methods

A single-center observational study was conducted at the largest HIV clinic in Tokyo, which treats 15% of the total patients in Japan, to determine the reasons for having diagnostic tests in newly infected individuals. HIV-infected patients who visited our clinic for the first time between 2011 and 2014 were analyzed.

Results

The 598 study patients comprised one-third of the total reported number of new patients in Tokyo during the study period. 76% were Japanese MSM. The reasons for being tested which led to the diagnosis was voluntary testing in 32%, existing diseases in 53% (AIDS-defining diseases in 22%, sexually transmitted infections (STI) in 8%, diseases other than AIDS or STIs in 23%) and routine pre-surgery or on admission screening in 15%. 52% and 74% of the study patients and patients presented with AIDS, respectively, had never been tested. The median CD4 count in patients with history of previous testing (315/μL) was significantly higher than that of patients who had never been tested (203/μL, p<0.001).

Conclusions

Only 32% of the newly HIV diagnosed patients were diagnosed because of voluntary testing, and 53% were diagnosed due to presence of other diseases. These results remain unchanged from our previous report 10 years earlier (2000–2004) on newly diagnosed patients at the same clinic. HIV testing has not been widely used by newly diagnosed patients in the Tokyo metropolitan area.     

Could Small Neurotoxins-Peptides be Expressed during SARS-CoV-2 Infection?

SARS-CoV-2 pathogenesis has been recently extended to human central nervous system (CNS), in addition to nasopharyngeal truck, eye, lung and gut. The recent literature highlights that some SARS-CoV-2 spike glycoprotein regions homologous to neurotoxin-like peptides might bind to human nicotinic Acetyl-Choline Receptors (nAChRs). Spike-nAChR interaction can probably cause dysregulation of CNS and cholinergic anti-inflammatory pathways and uncontrolled immune-response, both associated to a severe COVID-19 pathophysiology. Herein, we hypothesize that inside the Open Reading Frame (ORF) region of spike glycoprotein, the RNA polymerase can translate small neurotoxic peptides by means of a “jumping mechanism” already demonstrated in other coronaviruses. These small peptides can bind the snAChRs instead of Spike glycoproteins. A striking homology occurred between these small peptides observed by sequence retrieval and proteins alignment. Acting as nAChRs antagonists, these small peptides (conotoxins) could be the explanation for the extrapulmonary clinical manifestations (neurological, hemorrhagic and thrombotic expressions, the prolonged apnea, the cardiocirculatory collapse, the heart arrhythmias, the ventricular tachycardia, the body temperature alteration, the electrolyte K+ imbalance and finally the significant reduction of butyryl cholinesterase (BuChE) plasma levels, as observed in COVID-19 patients. Several factors might induce the expression of these small peptides, including microbiota. The main hypothesis regarding the presence of these small peptides opens a new scenario on the etiology of COVID-19 clinical symptoms observed so far, including the neurological manifestations.     

Etiology of Sarcoidosis: Does Infection Play a Role?

Sarcoidosis is a granulomatous inflammatory disorder of unclear etiology, which is known to affect multiple organ systems including the lungs, heart, skin, central nervous system, and eyes, among others. For this reason, sarcoidosis represents a systemic medical disorder that is clinically relevant to multiple medical sub-specialties. Despite extensive research, the etiology of sarcoidosis has yet to be elucidated, although most evidence supports that the pathogenetic mechanism of sarcoidosis is an aberrant immune response, driven by an unidentified antigen (or antigens) in genetically susceptible individuals. Multiple candidate etiologic agents, including microbial organisms and environmental agents, have been investigated, but study results are inconclusive. In this review, we describe the known histologic and immunologic features of sarcoidosis and discuss the evidence supporting a role for infectious processes in the pathogenesis of sarcoidosis.