29 May 2019

When a virus enters a biological fluid of a host—blood, lung, or cerebrospinal fluid—it becomes ensheathed by a unique set of proteins from its new environment. The proteins that make up the sheath differ depending on the fluid and on the type of virus. This “corona,” as Anna-Lena Spetz, Stockholm University, and Samir El Andaloussi at Stockholm’s Karolinska Institutet call the protein layer, shapes the interactions between virus and host cell. Besides rendering the virus more or less infectious, the corona incorporates amyloidogenic proteins. This changes their folding and spurs amyloid fibrils, which radiate from the viral surface, the authors report in the May 27 Nature Communications.

Viruses wrap themselves in a layer of host proteins.

The resulting “protein corona” makes a virus more or less infective.

When amyloid proteins are incorporated into the corona, fibrils radiate from the viral surface.

“We believe the mechanism we describe here can offer a physical explanation of the connection between viruses and amyloidopathies,” first author Kariem Ezzat told Alzforum. “Viral surfaces can induce conformational changes in amyloidogenic proteins, leading to a physical change from a soluble peptide to an insoluble one,” Ezzat said.

The corona seems to influence virus-host interactions, viral infectivity, and immune cell activation, according to Ruth Itzhaki, professor emerita at the University of Manchester, U.K. “This study provides further evidence that HSV1 plays a major role in AD,” she wrote to Alzforum. Itzhaki linked herpes simplex virus type 1 to Alzheimer’s almost 30 years ago (Jamieson et al., 1991).

Ezzat previously studied nanoparticles, which are known to collect a set of proteins on their surface that is unique to the fluid into which they are dunked (Monopoli et al., 2012). This corona becomes part of the particle’s identity and determines how it interacts with a cell (Cai and Chen, 2018; Walczyk et al., 2010).

Viruses, which are nano-sized and not metabolically active by themselves, interact with their hosts much like nanoparticles, Ezzat said. Viruses have been reported to interact with specific proteins, but Ezzat wanted to know whether they develop an entire corona. If they do, does the corona change depending on the virus, or the fluid it enters? And how might that influence host interactions?

To find out, Ezzat and colleagues incubated respiratory syncytial virus (RSV) and herpes simplex virus 1 (HSV-1) with different biological fluids in test tubes. They compared human plasma from adults and infants, human bronchoalveolar lavage fluid (BALF) from lungs, where RSV causes infection, and fetal bovine serum, which is often used in culture conditions. When the viruses were first produced in the lab without serum, their surfaces were relatively free of proteins (see image below). However, once incubated with a bodily fluid, each acquired a unique set of proteins on their outer envelope.

Protein Bath. RSV produced in serum-free conditions (left) has a bare outer envelope. Soaked in human plasma (middle) or fetal bovine serum (right), the virus becomes decorated with a layer of proteins (black arrowheads). [Courtesy of Ezzat et al., Nature Communications.]

The researchers used mass spectrometry to analyze which proteins stuck to RSV’s surface. Proteins incorporated into the viral corona were not the most abundant proteins in the fluid, suggesting they were selected based on viral surface properties. The composition of the corona differed for each virus and each fluid. For instance, in adult plasma, the corona of RSV consisted mostly of antibodies and complement factors, immune proteins that would neutralize the virus. By contrast, in BALF, RSV adsorbed proteins known to function in adhesion, anchoring, interspecies interaction, and parasitism—proteins that could make the virus more infective. This is consistent with the observation that RSV infects cells mainly in the lungs.

“Although the virus is the same on the genetic level no matter the microenvironment, it becomes a different entity based on the fingerprint of different proteins it acquires,” said Ezzat. “The proteins form a unique signature of a particular virus in a particular microenvironment.”

To see whether RSV’s corona changed its infectivity, the scientists used RSV that expressed green fluorescent protein, incubated it in a biological fluid, and infected human epithelial cells. Naked virus infected few cells, virus incubated in adult plasma infected even fewer, but RSV soaked in BALF infected more cells than naked virus. RSV that had acquired a corona from fetal bovine serum was the most infectious. Interestingly, RSV bathed in infant plasma also was more infective than naked virus, perhaps because babies lack the anti-RSV antibodies present in adult plasma.

What if the corona includes an amyloidogenic protein? Nanoparticles adsorb amyloidogenic proteins, which changes their conformation and concentrates them to catalyze aggregation (Gladytz et al., 2016; Cabaleiro-Lago et al., 2010). Do viruses do the same thing?

Sure enough, RSV catalyzed fibrillization of a peptide cut from the islet amyloid polypeptide, called NNFGAIL. A thioflavin-T assay, which indicates formation of amyloid fibrils, showed that fibrils of the peptide sprang from the surface of RSV particles, but were absent from a virus-free supernatant.

Fibril Generator. An HSV-1 particle (white arrowhead) catalyzes the formation of Aβ42 fibrils (black arrowheads). [Courtesy of Ezzat et al., Nature Communications.]

In the same fashion, HSV-1 catalyzed the fibrillization of Aβ42 within 100 minutes of co-incubation. The virus bound Aβ42 with higher affinity than Aβ40. In transmission electron microscope images, protofilaments and fibrils of Aβ42 appeared to contact the viral surface (image at left).

This virus-induced amyloid formation appeared to happen in vivo, as well. The researchers injected HSV-1 into the right lateral ventricles of 3-month-old 5XFAD mice, an age when Aβ pathology begins. They found more abundant Aβ42 accumulation in the hippocampus and cortex two days later, compared with transgenic mice injected with control supernatant (see image below).

The concept that viruses seed amyloid formation is not new. A study by Robert Moir and Rudolph Tanzi at Massachusetts General Hospital and co-authors showed HSV-1 and other human herpesviruses do it (Jun 2018 news). These authors report that this fibrillization protects mouse and cellular models of AD from infection, arguing that Aβ has an antimicrobial effect in the brain. “This interesting study nicely confirms these findings,” Tanzi wrote to Alzforum about Ezzat’s work. “The additional details described, regarding this process of nucleation of β-amyloid by viruses, could be helpful in devising therapeutic approaches for AD that prevent amyloid deposition driven by viruses and other microbes.”

Infection Increases Plaques. Three-month-old 5xFAD mice injected with HSV-1 (left) have more Aβ plaques (dots) than do mice injected with virus-free supernatant. [Courtesy of Ezzat et al., Nature Communications.]

Ezzat and colleagues noted that amyloid fibrils could make some viruses more infectious. They did not investigate the question but cited other work suggesting as much (Wojtowicz et al., 2002; Münch et al., 2007). However, Moir and Tanzi dispute this point in a separate comment to Alzforum, citing recent papers supporting an antimicrobial entrapment function for Aβ fibrillization (see comment below).

“It may be that corona proteins modulate stability or infectivity of viruses in a cellular setting, but it’s uncertain that they modulate in vivo infectivity. Many more experiments would be required to show this,” said Howard Federoff, University of California, Irvine. If intracranial infection with HSV-1 increases amyloid burden in 5XFAD mice, Federoff wondered whether this would be reflected in postmortem brain tissue of people who survived herpes encephalitis.

Regardless of the impact of amyloid fibrils, the authors suggest that the acquisition of a protein corona constitutes an important precellular phase of the viral life cycle, which until now was thought to begin once a virus contacts a cell. Ezzat acknowledges that examining two viruses is but a start, but he believes this could be a more general phenomenon. “It’s possible that all viruses accumulate an entire layer of factors that dramatically affects their pathogenesis,” he told Alzforum. The authors did not investigate if gene variants identified in AD GWAS affect the composition of a person’s viral corona, and influence subsequent consequences for infectivity or protein aggregation.

Could studying the corona improve vaccine design? Ezzat said that instead of using purified viruses in vaccine development, incorporating relevant coronal proteins could possibly make a more effective antigen.—Gwyneth Dickey Zakaib