A new type of enveloped virus?

All known virus particles can be placed into one of two general categories: enveloped or non-enveloped. Viruses that fall into the former category are characterized by a lipid membrane derived from the host cell, and one or more nuclecapsid proteins that interact with the viral genome. A virus that infects an archaeal host may constitute a new category of enveloped viruses. It comprises a membrane vesicle that encloses a circular ssDNA genome which is devoid of nucleic acid-binding nucleoproteins.

Examples of enveloped virions that contain nucleoproteins are shown in the figure below. These include influenza virus (left), a simple retrovirus (center), and a togavirus (right).

The influenza virion contains segments of viral RNA bound to four different proteins. Retroviral RNA is bound to a nucleocapsid protein which in turn is enclosed in a capsid, while togavirus RNA is located within an icosahedral shell.

Until recently, it was believed that the genome of all other known enveloped DNA and RNA viruses is always associated with one or more viral proteins. This belief may be changed by the isolation, from a solar saltern in Trapani, Italy, of a virus that infects the archaeal species Halorubrum. Salterns are multi-pond systems in which sea water is evaporated to produce salt. In such hypersaline envrionments, Archaea predominate, and about 20 archaeal viruses have been isolated from these locations.

The virus isolated from the Italian saltern is called Halorubrum pleomorphic virus-1, or HRPV-1. Biochemical analyses of the virion show that it is composed of lipids and two structural proteins, VP3 and VP5. The genome is a circular ssDNA about 7 kb in length with nine open reading frames. The virion architecture is unique: it is composed of a flexible membrane (hence the designation pleomorphic) that contains external spikes of the VP4 protein, and is lined on the interior with VP3. The viral DNA is apparently not bound to any proteins in the virions.

At the upper left is my depiction of the appearance of HRPV-1. The diagram was produced by deleting the internal proteins and nucleic acid of a simple retrovirus and replacing these with a ssDNA genome. The HRPV-1 VP4 spikes and the internal VP3 proteins are present, but no proteins are bound to the viral genome. Whether or not the VP4 spikes are oligomeric as shown is unknown.

Most enveloped viruses acquire their lipid membrane by budding from the host cell, and a similar mechanism could account for the formation of HRPV-1 virions. In the absence of a nucleoprotein, it is not clear how the viral genome would be specifically incorporated into the budding envelope. Another condundrum is how the virions would pass through the proteinaceous layer that covers the archaeal host cell.

Whether HRPV-1 is representative of a new kind of virus lacking nucleocapsid protein will be revealed by the study of other pleomorphic enveloped viruses. Candidates include bacterial viruses that infect mycoplasmas, and another pleomorphic haloarchaeal virus isolated from a different Italian saltern, Haloarcula hispanica pleomorphic virus 1.

Pietila, M., Laurinavicius, S., Sund, J., Roine, E., & Bamford, D. (2009). The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes Journal of Virology, 84 (2), 788-798 DOI: 10.1128/JVI.01347-09

Virology lecture #11: Assembly


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Packaging of the segmented influenza RNA genome

The RNA genome of influenza viruses is segmented . The virions of influenza A and B viruses contain 8 different RNAs, while those of influenza C viruses contain 7. How is the correct number of RNA segments inserted into newly synthesized virus particles?

During influenza virus assembly, viral RNAs and viral proteins – called a ribonucleoprotein complex or RNP –  travels to the plasma membrane. There the virion forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.

influenza-assembly

Production of an infectious virus particle requires incorporation of at least one copy of each of the eight RNA segments. Two different mechanisms – random and selective packaging – have been proposed to explain how each virion receives a full complement of genomic RNA.

If the 8 influenza viral RNA segments were randomly packaged into new particles, we would expect to observe 1 infectious particle for every 400 particles assembled (8!/88). This ratio falls within the range of infectious to noninfectious particles that occur in virus stocks. If more than 8 RNA segments could be packaged into each virion, then the fraction of infectious particles would be significantly increased. For example, if 12 RNA molecules could fit into each virion, then 10% of the particles would have the complete viral genome. In support of this mechanism, influenza viruses with more than 8 RNA segments have been observed.

In the selective packaging mechanism, each of the eight genomic RNAs has a different signal that allows incorporation into virus particles. These signals are believed to be within the noncoding and coding sequences at the 5′- and 3′-ends of the viral RNAs. The sequences interact and form structures that are unique to each segment, and which have been shown to be essential for incorporation of each segment into virions. Consistent with this hypothesis, electron microscopy reveals that during budding, the viral RNPs are organized in a distinct pattern, as shown in the image.

influenza-packaging

This observation argues that RNPs are not randomly incorporated into virions, and is consistent with the presence of specific signals in each RNA segment that enable the RNPs to be packaged as a complete set. The mechanisms by which these signals are recognized, and how they ensure incorporation of one copy of each RNA segment into the particle, are not known.

There is clear evidence for a selective mechanism during the packaging of the bacteriophage ψ6 genome. Viral particles contain one copy each of a S, M, and L dsRNA segment. All particles contain a complete complement of genome segments, as indicated by the fact that every virus particle is infectious. Only the S RNA segment can enter newly formed particles; once that segment is packaged, then the M RNA can enter. The L RNA can only enter particles that contain both the S and M segments. Precise packaging is therefore the result of a serial dependence of packaging of the RNA segments.

Muramoto, Y., Takada, A., Fujii, K., Noda, T., Iwatsuki-Horimoto, K., Watanabe, S., Horimoto, T., Kida, H., & Kawaoka, Y. (2006). Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions Journal of Virology, 80 (5), 2318-2325 DOI: 10.1128/JVI.80.5.2318-2325.2006

Noda, T., Sagara, H., Yen, A., Takada, A., Kida, H., Cheng, R., & Kawaoka, Y. (2006). Architecture of ribonucleoprotein complexes in influenza A virus particles Nature, 439 (7075), 490-492 DOI: 10.1038/nature04378

Frilander, M. (1995). In Vitro Packaging of the Single-stranded RNA Genomic Precursors of the Segmented Double-stranded RNA Bacteriophage ψ6: The Three Segments Modulate Each Other’s Packaging Efficiency Journal of Molecular Biology, 246 (3), 418-428 DOI: 10.1006/jmbi.1994.0096

Assembly of influenza virus

Our discussion of influenza virus replication has so far brought us to the stage of viral RNA synthesis. Last time we discussed the formation of viral RNAs, an event which takes place in the cell nucleus. Now we’ll consider how these RNAs participate in the assembly of new infectious viral particles, as illustrated in the following figure.

influenza-assembly

For simplicity, the nucleus is not shown. But remember that the viral RNAs have to be exported from the nucleus to the cytoplasm, where viral assembly occurs. First, the viral mRNAs are translated to produce all the proteins needed to synthesize a new virus particle. The mRNAs encoding the HA and NA glycoproteins are translated by ribosomes that are bound the the endoplasmic reticulum – the membranous organelle that assists in transporting certain proteins to the plasma membrane. As the HA and NA proteins are produced, they are inserted into the membrane of the endoplasmic reticulum as shown. These proteins are then transported to the cell surface via small vesicles that eventually fuse with the plasma membrane. As a result, the HA and NA are inserted in the correct direction in the lipid membrane of the cell. The M2 protein is sent to this location in a similar way.

The (-) strand viral RNAs that will be packaged into new virus particles are produced in the cell nucleus, then exported to the cytoplasm. These RNAs are joined with the viral proteins PA, PB1, PB2, and NP. Viral proteins other than HA, NA, and M2 are produced by translation on free ribosomes, as shown for M1. The latter protein binds to the membrane where HA, NA, and M2 have been inserted. The assembly consisting of viral RNAs and viral proteins – called a ribonucleoprotein complex or RNP –  travels to the site of assembly. The virion then forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.

As new virions are produced by budding, they would immediately bind to sialic acid receptors on the cell surface, were it not for the action of the viral NA glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. This requirement explains how the neuraminidase inhibitors Tamiflu and Relenza function: they prevent cleavage of sialic acid from the cell surface. In the presence of these inhibitors, virions bud from the cell surface, but they remain firmly attached. Therefore Tamiflu and Relenza block infection by preventing the spread of newly synthesized virus particles to other cells.

Ebola virus glycoprotein antagonizes tetherin

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The innate response to virus infection is a marvel: when confronted with an invading microbe, it responds rapidly by producing interferons and other cytokines which establish an antiviral state. Its effectiveness is underscored by the fact that every viral genome must encode countermeasures that modulate its activity. A recent paper in PNAS adds another mechanism to the evasion toolbox of Ebola virus.

The cellular protein tetherin is encoded by a gene whose transcription is induced by interferon, a so-called interferon-induced gene (ISG). The protein is found on the plasma membrane and in perinuclear compartments of the cell. Last year several groups demonstrated that the presence of tetherin in cells causes retention of retrovirus particles at the cell surface. The tethered particles are taken up into the cell, thereby reducing the yield of infectious virus. As would be expected, an HIV-1 protein – Vpu – inhibits the activity of tetherin. When Vpu is present, tetherin has no effect on the yield of HIV-1.

Because it is not possible to work with infectious Ebola virus except under strict containment, the authors instead studied virus-like particles (VLPs), which can be produced in cells by the synthesis of the viral VP40 protein. VLPs lack the viral genome and are not infectious. VLPs are readily produced in a cell line, 293T, which synthesizes very little tetherin. However, when tetherin was synthesized in 293T cells, the yield of VLPs was dramatically reduced, and the particles were retained at the cell surface.

The reduction of VLP yield by tetherin was overcome by synthesis of the Vpu protein of HIV-1. This finding prompted the authors to ask whether the genome of Ebola virus encodes a protein like Vpu that can antagonize tetherin restriction. They found that synthesis of the viral glycoprotein (GP) allowed VLP production in 293T cells in the presence of tetherin. Although the mechanism by which GP counteracts tetherin was not deciphered, the authors did find that GP binds tetherin directly. Furthermore, GP must remain anchored to the plasma membrane in order to exert its anti-tetherin activity.

It will be of great interest to determine how Ebola virus GP and proteins of other viruses permit virion budding from cells in the presence of tetherin. The results of such studies will not only contribute to our understanding of virion maturation, but could also suggest ways to produce a broad-spectrum inhibitor of enveloped viruses.

Kaletsky, R., Francica, J., Agrawal-Gamse, C., & Bates, P. (2009). Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein Proceedings of the National Academy of Sciences, 106 (8), 2886-2891 DOI: 10.1073/pnas.0811014106