Direct observation of membrane insertion by enveloped virus matrix proteins by phosphate displacement

Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes

Synthesis and antiviral activity of novel spirocyclic nucleosides

The synthesis of a number of spirocyclic ribonucleosides containing either a triazolic or azetidinic system is described, along with two analogous phosphonate derivatives of the former. These systems were constructed from the same β-D-psicofuranose starting material. The triazole spirocyclic nucleosides were constructed using the 1-azido-1-hydroxymethyl derived sugars, where the primary alcohol was alkylated with a range of propargyl bromides, whereas the azetidine systems orginated from the corresponding 1-cyano-1-hydroxymethyl sugars. Owing to their close similarity with ribavirin, the library of compounds were investigated for their antiviral properties using MHV (Murine Hepatitis Virus) as a model

Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None of these viruses was found to be significantly less fit than wild-type, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane-associated replication complexes and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication

Lipid phosphate is displaced in the presence of viral matrix proteins.

<p>Relative electron microscope signal intensity is shown on the vertical axis with average background intensity marked by a gray triangle. The horizontal axis represents radial distance from the midpoint of the membrane. The boundaries of the inner (In) and outer (Out) membrane phosphate rings measured in this study are shown for viruses (black), empty vesicles (blue) and virus-like particles that contain surface glycoproteins but lack a visible matrix layer (GP vesicles; red). Approximate positions of the nucleoprotein (core; 3MX5), matrix (2KO5) and glycoprotein (3KAS) structures in arenavirus particles are shown as a reference. P-values relate to comparison of inner phosphate ring signals with viruses as described in the Methods section. Comparisons are omitted where GP vesicles were not available. Virus names are abbreviated as follows: <i>Lymphocytic choriomeningitis virus</i> (LCMV), <i>Junin virus</i> (JUNV), <i>Pichinde virus</i> (PICV), <i>Tacaribe virus</i> (TCRV), <i>Porcine respiratory and reproductive syndrome virus</i> (PRRSV), <i>Feline coronavirus</i> (FCoV), <i>Mouse hepatitis virus</i> (MHV), <i>Severe acute respiratory syndrome coronavirus</i> (SARS-CoV), <i>Influenza A virus</i> (FLUAV), <i>Influenza B virus</i> (FLUBV), <i>Murine leukaemia virus</i> (MLV).</p

Apparent electron density is constant for small and large virus particles.

<p>The virion edge was sampled at four positions described in Fig. 3 (In, Core, Matrix, Glycoprotein) and at the background ice beyond the Glycoprotein (Ice). Each datapoint shows the average density for 8 samples from 12 <i>Tacaribe virus</i> particles of similar size. Error bars indicate standard deviation.</p

Presence of matrix proteins at the virion surface is necessary to maintain elongated virus shapes.

<p>(A) Shape and perimeter length are shown for 252 <i>Influenza A virus</i> particles and 66 GP vesicles. Coefficients of determination and statistical confidence measures are shown for virus particles and GP vesicles of <i>Lymphocytic choriomeningitis virus</i> (LCMV), <i>Tacaribe virus</i> (TCRV) and <i>Influenza A virus</i> (FLUAV), and for empty vesicles of cellular origin (B).</p

Cryo-electron micrographs of virus preparations.

<p>The images include virus particles (v), GP vesicles (g), empty vesicles (e) and tubular hollow particles (t). Preparations of <i>Tacaribe virus</i> (A), <i>Porcine respiratory and reproductive syndrome virus</i> (B), <i>Severe acute respiratory syndrome coronavirus</i> (C) and <i>Influenza A virus</i> (D) are shown to illustrate the double-ringed appearance of the membrane.</p