Faculty Opinions recommendation of Tracking viral genomes in host cells at single-molecule resolution.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

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Bacteriophage self-counting in the presence of viral replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104163118 ◽

2021 ◽

Vol 118 (51) ◽

pp. e2104163118

Author(s):

Tianyou Yao ◽

Seth Coleman ◽

Thu Vu Phuc Nguyen ◽

Ido Golding ◽

Oleg A. Igoshin

Keyword(s):

Gene Expression ◽

Viral Replication ◽

Viral Gene Expression ◽

Host Cells ◽

Optimal Decision ◽

Viral Gene ◽

Viral Genomes ◽

Viral Copy Number ◽

Viral Copy ◽

Type Phage

When host cells are in low abundance, temperate bacteriophages opt for dormant (lysogenic) infection. Phage lambda implements this strategy by increasing the frequency of lysogeny at higher multiplicity of infection (MOI). However, it remains unclear how the phage reliably counts infecting viral genomes even as their intracellular number increases because of replication. By combining theoretical modeling with single-cell measurements of viral copy number and gene expression, we find that instead of hindering lambda’s decision, replication facilitates it. In a nonreplicating mutant, viral gene expression simply scales with MOI rather than diverging into lytic (virulent) and lysogenic trajectories. A similar pattern is followed during early infection by wild-type phage. However, later in the infection, the modulation of viral replication by the decision genes amplifies the initially modest gene expression differences into divergent trajectories. Replication thus ensures the optimal decision—lysis upon single-phage infection and lysogeny at higher MOI.

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Monitoring Early Fusion Dynamics of Human Immunodeficiency Virus Type 1 at Single-Molecule Resolution

Journal of Virology ◽

10.1128/jvi.00053-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 7022-7033 ◽

Cited By ~ 38

Author(s):

Terrence M. Dobrowsky ◽

Yan Zhou ◽

Sean X. Sun ◽

Robert F. Siliciano ◽

Denis Wirtz

Keyword(s):

Human Immunodeficiency Virus ◽

Tensile Strength ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Single Molecule ◽

Viral Envelope ◽

Host Cells ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The fusion of human immunodeficiency virus type 1 (HIV-1) to host cells is a dynamic process governed by the interaction between glycoproteins on the viral envelope and the major receptor, CD4, and coreceptor on the surface of the cell. How these receptors organize at the virion-cell interface to promote a fusion-competent site is not well understood. Using single-molecule force spectroscopy, we map the tensile strengths, lifetimes, and energy barriers of individual intermolecular bonds between CCR5-tropic HIV-1 gp120 and its receptors CD4 and CCR5 or CXCR4 as a function of the interaction time with the cell. According to the Bell model, at short times of contact between cell and virion, the gp120-CD4 bond is able to withstand forces up to 35 pN and has an initial lifetime of 0.27 s and an intermolecular length of interaction of 0.34 nm. The initial bond also has an energy barrier of 6.7 kB T (where kB is Boltzmann's constant and T is absolute temperature). However, within 0.3 s, individual gp120-CD4 bonds undergo rapid destabilization accompanied by a shortened lifetime and a lowered tensile strength. This destabilization is significantly enhanced by the coreceptor CCR5, not by CXCR4 or fusion inhibitors, which suggests that it is directly related to a conformational change in the gp120-CD4 bond. These measurements highlight the instability and low tensile strength of gp120-receptor bonds, uncover a synergistic role for CCR5 in the progression of the gp120-CD4 bond, and suggest that the cell-virus adhesion complex is functionally arranged about a long-lived gp120-coreceptor bond.

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Viral Genome Conformations and Contacts across Different Lifecycle Stages

Proceedings ◽

10.3390/proceedings2020050109 ◽

2020 ◽

Vol 50 (1) ◽

pp. 109

Author(s):

Peter G. Stockley ◽

Nikesh Patel ◽

Emma L. Wroblewski ◽

Andrew J. P. Scott ◽

Carlos P. Mata ◽

...

Keyword(s):

Conformational Changes ◽

Drug Targets ◽

Three Dimensional ◽

Host Cells ◽

Dimensional Structure ◽

Viral Genomes ◽

Starting Point ◽

B Virus ◽

Infection Mechanisms ◽

Conformational Memory

Single-stranded RNA viral genomes (gRNA) are dynamic molecules that permit packaging into virions and their subsequent extrusion during infection. For viruses with such genomes, we discovered a previously unsuspected mechanism that regulates their assembly. This regulation is the result of multiple cognate coat protein (CP)–gRNA contacts distributed across the RNA. Collectively, these interactions make the assembly highly efficient and specific. The regions of the gRNA packaging signals (PSs) driving this assembly are potential drug targets, whilst the manipulation of PS–CP contacts with nonviral RNA cargos is a route towards bespoke virus-like particles. Infectivity depends on the virions being able to transfer their gRNAs into host cells. The starting point for this transfer appears to be an encapsidated RNA with a defined three-dimensional structure, especially around the PSs. A combination of asymmetric cryo-electron microscopy structure determination and X-ray synchrotron footprinting were used to define these contacts and structures in a number of viral examples, including hepatitis B virus and enteroviruses. These tools allow us to look beyond the outer CP layer of the virion shell and to see the functional, asymmetric components that regulate viral infectivity. This revealed yet more unexpected aspects of critical infection mechanisms, such as the RNA conformational changes required for encapsidation, the details of PS–CP contacts regulating the assembly, and the conformational “memory” imposed by encapsidation.

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Bacteria facilitate viral co-infection of mammalian cells and promote genetic recombination

10.1101/154021 ◽

2017 ◽

Cited By ~ 2

Author(s):

A.K. Erickson ◽

P.R. Jesudhasan ◽

M.J. Mayer ◽

A. Narbad ◽

S.E. Winter ◽

...

Keyword(s):

Mammalian Cells ◽

Genetic Recombination ◽

Enteric Viruses ◽

Intestinal Bacteria ◽

Enteric Virus ◽

Viral Fitness ◽

Host Cells ◽

Bacterial Strains ◽

Viral Recombination ◽

Viral Genomes

SUMMARYIntestinal bacteria promote infection of several mammalian enteric viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact enteric viruses. We found that most bacterial strains bound poliovirus, a model enteric virus. Given that each bacterium bound multiple virions, we hypothesized that bacteria may deliver multiple viral genomes to a mammalian cell even when very few virions are present, such as during the first replication cycle after inter-host transmission. We found that exposure to certain bacterial strains increased viral co-infection even when the ratio of virus to host cells was low. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated viral fitness restoration through genetic recombination. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections.

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MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

10.1101/2021.01.27.428469 ◽

2021 ◽

Author(s):

CM Gallardo ◽

S Wang ◽

DJ Montiel-Garcia ◽

SJ Little ◽

DM Smith ◽

...

Keyword(s):

Long Range ◽

Single Molecule ◽

Vaccine Development ◽

Complex Mixture ◽

Therapeutic Benefit ◽

Read Length ◽

Clinical Samples ◽

Viral Genomes ◽

Immune Pressure ◽

Sequencing Platforms

AbstractTechnical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

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Lipid-Specific Labeling of Enveloped Viruses with Quantum Dots for Single-Virus Tracking

mBio ◽

10.1128/mbio.00135-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Li-Juan Zhang ◽

Shaobo Wang ◽

Li Xia ◽

Cheng Lv ◽

Hong-Wu Tang ◽

...

Keyword(s):

Quantum Dots ◽

Optical Properties ◽

Virus Infection ◽

Quantum Dot ◽

Single Molecule ◽

Host Cells ◽

Single Molecule Tracking ◽

Enveloped Viruses ◽

Single Virus Tracking ◽

Labeling Method

ABSTRACT Quantum dots (QDs) possess optical properties of superbright fluorescence, excellent photostability, narrow emission spectra, and optional colors. Labeled with QDs, single molecules/viruses can be rapidly and continuously imaged for a long time, providing more detailed information than when labeled with other fluorophores. While they are widely used to label proteins in single-molecule-tracking studies, QDs have rarely been used to study virus infection, mainly due to a lack of accepted labeling strategies. Here, we report a general method to mildly and readily label enveloped viruses with QDs. Lipid-biotin conjugates were used to recognize and mark viral lipid membranes, and streptavidin-QD conjugates were used to light them up. Such a method allowed enveloped viruses to be labeled in 2 h with specificity and efficiency up to 99% and 98%, respectively. The intact morphology and the native infectivity of viruses were preserved. With the aid of this QD labeling method, we lit wild-type and mutant Japanese encephalitis viruses up, tracked their infection in living Vero cells, and found that H144A and Q258A substitutions in the envelope protein did not affect the virus intracellular trafficking. The lipid-specific QD labeling method described in this study provides a handy and practical tool to readily “see” the viruses and follow their infection, facilitating the widespread use of single-virus tracking and the uncovering of complex infection mechanisms. IMPORTANCE Virus infection in host cells is a complex process comprising a large number of dynamic molecular events. Single-virus tracking is a versatile technique to study these events. To perform this technique, viruses must be fluorescently labeled to be visible to fluorescence microscopes. The quantum dot is a kind of fluorescent tag that has many unique optical properties. It has been widely used to label proteins in single-molecule-tracking studies but rarely used to study virus infection, mainly due to the lack of an accepted labeling method. In this study, we developed a lipid-specific method to readily, mildly, specifically, and efficiently label enveloped viruses with quantum dots by recognizing viral envelope lipids with lipid-biotin conjugates and recognizing these lipid-biotin conjugates with streptavidin-quantum dot conjugates. It is not only applicable to normal viruses, but also competent to label the key protein-mutated viruses and the inactivated highly virulent viruses, providing a powerful tool for single-virus tracking.

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Novel Protocol for Estimating Viruses Specifically Infecting the Marine Planktonic Diatoms

Diversity ◽

10.3390/d12060225 ◽

2020 ◽

Vol 12 (6) ◽

pp. 225

Author(s):

Yuji Tomaru ◽

Kei Kimura

Keyword(s):

Culture Method ◽

Host Cells ◽

Natural Sediment ◽

Viral Genomes ◽

Planktonic Diatoms ◽

Ecological Relationships ◽

Ssrna Virus ◽

Single Host ◽

Complete Lysis ◽

Ssdna Viruses

Since their discovery, at least 15 diatom viruses have been isolated and characterised using a culture method with two cycles of extinction dilution. However, the method is time consuming and laborious, and it isolates only the most dominant virus in a water sample. Recent studies have suggested inter-species host specificity of diatom viruses. Here, we describe a new protocol to estimate previously unrecognised host-virus relationships. Host cell cultures after inoculation of natural sediment pore water samples were obtained before complete lysis. The proliferated viral genomes in the host cells were amplified using degenerate primer pairs targeting protein replication regions of single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) viruses, and then sequenced. Diverse ssRNA virus types within known diatom virus group were detected from inoculated Chaetoceros tenuissimus and C. setoensis cells. A previously unknown ssDNA virus type was detected in inoculated C. tenuissimus cells, but not in C. setoensis cells. Despite the possible protocol biases, for example non-specific adsorptions of virions onto the host cells, the present method helps to estimate the viruses infectious to a single host species. Further improvements to this protocol targeting the proliferated viral genomes might reveal unexpected diatom–virus ecological relationships.

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Cell-to-cell and genome-to-genome variability of adenovirus transcription tuned by the cell cycle

Journal of Cell Science ◽

10.1242/jcs.252544 ◽

2020 ◽

Vol 134 (5) ◽

pp. jcs252544

Author(s):

Maarit Suomalainen ◽

Vibhu Prasad ◽

Abhilash Kannan ◽

Urs F. Greber

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Single Molecule ◽

Click Reaction ◽

Viral Gene Expression ◽

Early Gene ◽

Viral Gene ◽

Viral Genomes ◽

E1a Gene ◽

G1 Cells

ABSTRACTIn clonal cultures, not all cells are equally susceptible to virus infection, and the mechanisms underlying this are poorly understood. Here, we developed image-based single-cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts using single-molecule RNA fluorescence in situ hybridization (FISH) and the incoming 5-ethynyl-2′-deoxycytidine (EdC)-tagged viral genomes using a copper(I)-catalyzed azide–alkyne cycloaddition (click) reaction. Surprisingly, expression of the immediate early gene E1A only moderately correlated with the number of viral genomes in the cell nucleus. Intranuclear genome-to-genome heterogeneity was found at the level of viral transcription and, in accordance, individual genomes exhibited heterogeneous replication activity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A gene expression and displayed increased correlation between E1A gene expression and viral genome copy numbers. The combined image-based single-molecule procedures described here are ideally suited to explore the cell-to-cell variability in viral gene expression in a range of different settings, including the innate immune response.

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