Category Archives: Molecular Virology

Can miRNAs help further attenuate influenza A vaccines?

Contributed by Guest Blogger: Brittany Sider ’11

MicroRNA (miRNA) molecules, first characterized in the early 1990s, have been implicated in a variety of different biological mechanisms. It took almost a decade for researchers to detect and understand the role of miRNAs in regulation of translation. Since then, research has focused on how we can scientifically manipulate these regulating molecules to our advantage in order to further understand biological underpinnings of certain diseases, as well as potential miRNA-based therapies.

The ability of the influenza virus to undergo frequent and substantial genomic mutations forces us to continually monitor its prevalence, and modify yearly vaccines to target the prevailing viral strains. Recently, live attenuated influenza vaccines (LAIVs, e.g. FluMist) have been proven effective, and have been distributed to a large portion of the eligible population to combat the seasonal flu. These vaccines are manipulated to become much more temperature-sensitive, and therefore are only capable of replicating in temperatures found in the nose. The inability of these attenuated viruses to replicate in the respiratory tract (due to higher temperatures) allows the vaccinated individual to produce antibodies to the influenza strains in the vaccine from the infection in the nasal passage. Therefore, the individual can produce the correct immune response without the virus spreading to the respiratory tract and causing symptoms.

In 2009, a group of researchers from Mount Sinai School of Medicine found that using microRNA response elements (MREs) can supplement the effectiveness of LAIVs. In the study, the MREs for the miR-124 (neural tissue-specific) and miR-93 (a ubiquitous miRNA) were inserted into open reading frames of influenza A nucleoprotein coding regions. The investigators vaccinated mice with miR-93-seeded strains, and then inoculated them with a lethal dose of influenza A/PR/8/34 H1N1 21 days later. This resulted in 100% survival of the subjects, as well as a robust immune response. In an attempt to attribute these results to other influenza strains, the same experiment was done with H5N1 (MREs were inserted into the vaccine specific for H5N1, and methods were repeated). Subjects who had received mock vaccinations 21 days prior to being inoculated with H5N1 displayed rapid weight loss, as well as 100% mortality. On the other hand, mice that had received the MRE-containing H5N1 strain did not display any signs of disease. Furthermore, serum from these subjects exhibited neutralizing activity against the wild-type H5N1, and a wide array of antibody responses (high levels of IgM, IgG1, IgG2a and IgG2b).

The results from this study lead the researchers to believe that MRE-containing LAIVs can be used, and potentially be even more effective than currently available LAIVs in protecting against influenza A outbreaks. In addition, this technology provides the potential to control for the degree of attenuation of the vaccine by manipulating the number of MREs/miRNAs. Lastly, FluMist – although proven to be equally as effective as injected vaccines – has some age exclusions. Perhaps the addition of MREs/miRNAs could expand the target demographic of this method of vaccination.


A different alphabet, a different treatment?

Contributed by Guest Blogger: Sean Koerner ’11

It’s easy to think of viruses as alien or lifeless – after all, they can’t reproduce on their own, eat anything, or even move around without assistance. However, viruses have evolved to use the same toolbox that human cells use, right down to the way their genes and proteins are encoded. One of the most problematic viruses for humans, HIV, works by putting its own information into our cells’ genomes, turning host cells into viral factories. This information is formed from two types of alphabets: strung-together sequences of deoxyribonucleotides, which exist intracellularly as deoxyribonucleotide triphosphate (dNTP) monomers in our own cells and ribonucletides, which form the HIV genome as well as existing independently as ribonucleotide triphosphate (rNTP) monomers within our own cells. In order to infect our cells, HIV uses a protein known as reverse transcriptase to generate the DNA that our cells are used to reading from the viral RNA genome. This reverse transcription of RNA to DNA has long been a target of anti-HIV drugs, since without this step, HIV cannot successfully infect our cells.

Recently, a team at the University of Rochester discovered a previously unknown characteristic of this process. Two of the cells most commonly infected by HIV, CD4+ lymphocytes and macrophages, displayed different levels of dNTPs and rNTPs after being infected by HIV, with the lymphocytes containing much less rNTPs and more dNTPs than the macrophages. After a biochemical analysis of the cells, the research team discovered that HIV’s reverse transcriptase is capable of using cellular rNTPs to generate RNA based upon the HIV genome, which is then reverse transcribed into cellular DNA while in the macrophage environment. This allows HIV to use the higher concentrations of rNTPs in macrophages to continue replicating efficiently, despite the relative dearth of dNTPs as compared to lymphocytes. Since HIV uses one method (dNTPs) in lymphocytes and one method (rNTPs) in macrophages, it may be possible to target HIV replication in macrophages specifically. Why care about the difference between the two cell types? Well, macrophages travel the body much more rapidly than lymphocytes; if we can stop HIV infecting them, we may be able to slow the progression of HIV infection throughout the body.

How could we do that? In short, by targeting the synthesis of rNTP strands with new drugs. Although we would likely experience side effects, they could be negligible compared with the repression of HIV. The research team at Rochester have already demonstrated that rNTP string inhibitors slow HIV’s infection of macrophages, so specific drugs targeted for this process might be able to halt it altogether.


Curing infected cells instead of killing them

Once a cell is infected, our immune system clears the infection not by targeting the virus, but by simply killing the infected cell.  Antibodies are also an important part of our defense against viral infections, but their function is limited to targeting viruses floating around outside the cell.  It is believed the once the virus enters the cell, it’s safe from antibodies.  But a recent study suggests that this may not be true; it seems that antibodies can eliminate viruses from inside cells too.  This is particularly groundbreaking because this may be a mechanism to cure infected cells, rather than just killing them.

Researchers identified a protein called TRIM21 that binds antibodies, but it was localized to the cell’s cytoplasm.  Why would it be there, when antibodies are secreted?  It seems that sometimes viruses can enter a cell with antibodies bound to it.  While some antibodies are neutralizing, meaning that they prevent attachment to the receptor, others are not.  If the virus is coated with these non-neutralizing antibodies, it can still enter, and it bring the antibodies in with it.  TRIM21 recognizes these internalized antibodies, with virions attached to them, and then targets it for destruction in a cellular blender called the proteasome. In an appropriately named “fate of capsid” experiment, the researchers showed that antibody bound adenovirus capsid proteins were being degraded as soon as 2 hours after infection and that the degradation required both the proteasome and TRIM21.  So the virus is being destroyed quickly, before it gets the chance to replicate.

As always, this study introduces many new questions.  They used adenovirus, which causes mild respiratory infections, but does the mechanism extend to other non-enveloped viruses? Can this mechanism be used to improve existing vaccines or develop new ones? How important is this mechanism in the overall response to viral infection?  Have viruses evolved mechanisms to block TRIM21?


1918 and 2009

The 1918 influenza pandemic (the “Spanish Flu”), by some estimates, killed as many as 100 million people in a very short period of time.  The 2009 “Swine Flu” pandemic didnt kill so many, but it spread rapidly and widely across the globe.  Despite that difference, it turns out the two viruses responsible for these pandemics have some important similarities.

Influenza virus has a protein on the surface called hemagglutinin, or HA, which is used to attach to host cells, allowing the virus to then enter and replicate.  HAs change rapidly, which is partly why influenza keeps coming back.  When HA changes, your antibodies dont recognize it so well, so you get sick again.  It turns out that the HAs of 2009 and 1918 are similar on both the sequence and structure level.  There is a small patch on the HA protein that is 95% identical between 1918 and 2009 but only 70% identical to seasonal strains.  Looking only at the 3D structure, among all influenza HAs, the 2009 HA is most similar to the 1918 HA.  The 1918 and 2009 HAs also lack glycosylation at the tip, while seasonal influenza viruses HAs are sugary.

Why is that interesting? An unusual pattern was noted in the 2009 pandemic: elderly people were not as affected as younger people, the reverse of what is usually seen with influenza.  It was proposed that perhaps some people still had immunity to the 1918 virus, which continued to circulate for many years after 1918, and that immunity was cross-protective.  A recent study shows that this indeed seems to be the case.  Mice immunized with the 1918 virus are protected against the 2009 virus.  The converse is also true: if you immunize mice with the 2009 virus, they are protected against the 1918 virus.  That’s pretty impressive when you consider that one season’s vaccine might not protect you from next season’s virus. It seems the immune system cant really tell the difference between these viruses.  Note that it also tells us how long immunity can last!  The next question is, how and why has this HA structure come back?


Ebola Virus Entry

Viruses can enter cells through a variety of different pathways.  Many enter through endocytosis, and there are actually several endocytic pathways: clathrin mediated, caveolin mediated, phago- and pinocytosis, and the rather mysterious “non-clathrin, non-caveolin mediated endocytosis.”

Ebola virus causes a severe hemorrhagic disease with 90% mortality.  Its an obviously frightening virus which makes it difficult to study, but knowing the details of its replication cycle may provide important clues on how to treat or prevent the disease.  A recent paper demonstrates that Ebola probably uses clathrin-mediated endocytosis.  Clathrin is a protein that forms a polyhedral lattice on the inside of the cell membrane helping to form vesicles.  Virus attachment induces this vesicle formation, giving the virus access to the cell by entering through these vesicles.  Among other experiments, they found that if you use the drug chorpromazine, which inhibits clathrin function, you can block Ebola entry.

The paper raises some interesting questions. First, they didnt actually use Ebola virus.  They used a modified HIV that expresses the Ebola virus glycoprotein involved in attachment and entry.  Does the natural virus enter in the same way?  They used several different cells in culture and found clathrin dependence in all of them, but is it the same in an infected animal?  Finally is the drug chlorpromazine one that could be used clinically?  Presumably not since disrupting clathrin mediated endocytosis would probably have a broadly toxic effect on the host, but it is an interesting lead compound.


Distant Evolutionary Relationships

We’ve been talking about protein structure and folding in my Biol 105 class.  Proteins are made of chains of amino acids and the sequence of amino acids, or primary structure, dictates the way the protein will fold into its final 3D or tertiary structure.  We may assume that two proteins with similar sequences would have a similar structure, and that two proteins with very different sequences would have different structures.  However, this is not true.  Proteins with very different sequences can end up with similar 3D structures.

A great example of this is the structure of capsid proteins from three very different viruses.  Adenoviruses infect animals (eukaryotes), and is one of many viruses that cause colds.  PRD1 is a bacteriphage, a virus that infects bacteria.  STIV (Sulfolobus turreted icosahedral virus) infects Sulfolobus, an archaea that lives in geothermal hotsprings in Yellowstone National Park.  STIV and its host love the 80 degree celsius, pH 3 environment of the hotsprings.  The fact that there are viruses that infect archaea in those extreme environments is cool enough.  But it turns out that the capsid proteins of these three viruses are actually quite similar.  Their sequence differs significantly, but their tertiary structures are highly similar, meaning these very different polypeptides fold into essentially the same shape.

What is the basis of this similarity?  Do all theses viruses share a common ancestor, which would have existed before the three domains of cellular life (eukarya, bacteria, archaea) diverged over 3 billion years ago? Is it convergent evolution?  Was there a horizontal gene transfer event in which a gene moved among all three domains?  The authors of the paper argue for a common ancestor but the other possibilities have not been formally excluded.  We still don’t really know, and it raises interesting questions about the origin of viruses.


Sputnik: Hijacking the Big Mama

A question I often get in class is: “Are there viruses that infect other viruses?”

The answer is still “no” but a recent discovery reported in Nature does starts to blur that line.  A newly identified virus, called Sputnik, tags along with another virus called Mamavirus (so called because its bigger than the previous record holder for large viruses, Mimivirus).

Interestingly, Sputnik virions can be found within Mamavirus virions, so they travel together. Since Sputnik can not replicate with in the Mamavirus virion, this is not an active infection of Mamavirus, but rather it is a passive particle traveling within the Mamavirus virion.

Importantly, Sputnik is a parasite of Mimivirus.   It can only replicate in ameoba that are co-infected by Mamavirus.  Parasites live off of other organisms, with a deleterious effect on the host, such as reduced nutrient uptake or growth rate.  Viruses are parasites – infected cells usually halt all or most cellular activity and eventually die, the most deleterious effect of all.  Sputnik is a parasite of Mamavirus it replicates at the expense of Mamavirus.  Cells infected with both viruses produce less 70% less infectious Mamavirus particles than a cell not co-infected with Sputnik.  A few other viruses have been identified that require co-infection with another virus (Hepatitis D virus for example) but these do not reduce the infectivity of the host virus so are not really parasites of the other virus.

Mamavirus hijacks a cell to replicate.  Sputnik hijacks Mamavirus for transportation and its cell-hijacking capabilities.  It is the first described virus of a new group of viruses called virophages.