Did viruses enable sex?

Dengue virus E glycoproteins (monomer at top) lie flat on the virus particle as dimers (middle). At endosomal low pH, three monomers reorient to place the fusion peptide (orange) into the cell membrane. Image credit.

A key step in sexual reproduction is the fusion of haploid cells to form a diploid zygote, yet the molecular mechanism underlying this joining of cells is poorly understood. Two studies reveal amazing similarities between proteins required for fusion of sperm and egg, and virus with host cells.

A screen for genes that cause male sterility in the flowering plant Arabidopsis led to the identification of the HAP2 protein. This protein was later found to be important for sperm-egg fusion in Arabidopsis and in the unicellular algae Chlamyodomonas. 

Homology modeling shows that the HAP2 protein looks very much like a class II viral fusion protein (illustrated). Found in dengue virus and many related viruses, dimers of these viral glycoproteins lie flat on the viral membrane, and are comprised largely of beta-strands. At one end of the protein is a fusion loop which allows the virus and cell membranes to join at the start of infection.

The HAP2 protein also has what looks to be a viral fusion loop. Removal or alteration of this sequence in Tetrahymena prevents fusion of mating cells. The fusion loop of the dengue virus E glycoprotein cannot substitute for the HAP2 sequence. Furthermore, vesicular stomatitis viruses with HAP2 in place of the viral glycoprotein cannot enter cells. However the results of biophysical experiments indicate that the HAP2 fusion loop can interact with membrane lipids in ways reminiscent of viral fusion peptides.

Solution of the atomic structure of HAP2 reveals a trimer with protein folds and an upright ‘hairpin’ configuration (illustrated for dengue virus) typical of class II fusion proteins. While acidification of viral type II fusion proteins is required for rearrangement to the post-fusion form, the trigger for HAP2 is not known.

These results clearly show that HAP2 is a type II fusion protein that mediates the joining of haploid gametes in the first step of sexual reproduction. These viral and cell proteins are so similar that it is highly improbable that they arose by convergent evolution. HAP2 is ancient: besides green algae and plants, it is also found in unicellular protozoa, cnidarians, hemichordates, and arthropods, indicating that it was likely present in the last common ancestor of eukaryotes. But viruses existed before the evolution of eukaryotic sex, raising the scenario that type II fusion proteins first arose in viruses, which provided them to eukaryotic cells for use in gamete cell fusion.

Without viruses, there would be no sex, and therefore no humans, or many other animals on Earth.

We continue to recognize new ways that the evolution of eukaryotic life has depended on viruses. These include a viral gene used to produce the placenta; enhancer elements for innate immunity; prions; and the nucleus. What exactly did eukaryotes invent?

TWiV 431: Niemann-Pick of the weak

The TWiVirions reveal bacteriophage genes that control eukaryotic reproduction, and the biochemical basis for increased Ebolavirus glycoprotein activity during the recent outbreak.

You can find TWiV #431 at microbe.tv/twiv, or listen below.

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A virus that controls reproduction

WolbachiaThe obligate intracellular bacteria Wolbachia (pictured), which infects 40% of arthropods, can manipulate its host to ensure its maintenance in the population. An example is cytoplasmic incompatibility, which occurs when infected males mate with uninfected females, and causes embryonic lethality (mating with an infected female produces viable offspring). Two Wolbachia genes responsible for this phenotype have been identified, and they are viral (link to paper).

A comparison of genome sequences of different Wolbachia strains that do or do not cause cytoplasmic incompatibility (CI) revealed two genes that were candidates for this phenotype. Both genes are transcribed in the testes of fruit flies, but at lower levels in older male flies which show decreased CI.

When either gene was expressed in male transgenic fruit flies, there was no effect on hatch rates after mating with uninfected females. When both genes were expressed in male flies, mating with uninfected females led to substantially reduced hatch rates. This transgene-induced lethality was rescued when the flies were mated with Wolbachia-infected females.

The two genes that together cause CI are called cytoplasmic incompatibility factor A and B (cifA, cifB). The cytological defects caused by these genes resemble those observed in Wolbachia-induced CI: most embryos do not divide more than two or three times.

Remarkably (or perhaps not!), cifA and cifB are not Wolbachia genes, but are viral. Wolbachia are infected with a bacteriophage called WO; nearly all sequenced Wolbachia genomes contain integrated WO DNA, and it is within this WO prophage that are found cifA and cifB. In other words, the ability of Wolbachia to control the reproduction of its arthropod host is regulated by two viral genes integrated in the bacterial genome.

Because CI caused by Wolbachia is a means of increasing their proportion in the female line (the bacteria are maternally inherited), cifA and cifB also enable spread of WO bacteriophage.

How cifA and cifB cause CI is unknown – most of the encoded proteins have no recognized protein domains with the exception of weak homology to proteases.  Understanding this mechanism might also contribute to controlling the spread of arboviruses: Wolbachia is known to inhibit replication of some mosquito borne viruses such as dengue virus and Zika virus.

TWiV 430: The persistence of herpesvirus

The TWiX cabal discuss sexual transmission of Zika virus in mice, and how immune escape enables herpes simplex virus escape from latency.

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TWiV 429: Zika Experimental Science Team

Vincent meets with members of team ZEST at the University of Wisconsin Madison to discuss their macaque model for Zika virus pathogenesis.

You can find TWiV #429 at microbe.tv/twiv, or listen/watch right here.

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Viral RNA is not infectious virus!

Zika RNA and virusA study of sexual transmission of Zika virus among mice (link to paper) demonstrates beautifully that viral nucleic acid detected by polymerase chain reaction (PCR) is not the same as infectious virus.

Male mice were infected with Zika virus and then mated with female mice. Efficient sexual transmission of the virus from males to females was observed. This observation in itself is very interesting but is not the focus of  my comments.

To understand the dynamics of sexual transmission, the authors measured Zika virus shedding in seminal fluid – by both PCR, to detect viral RNA, and by plaque assay, to detect infectious virus. The results are surprising (see figure – drawn in my hotel room).

Zika virus RNA persisted in semen for up to 60 days – far longer than did infectious virus, which could not be detected after about three weeks.

Many laboratories choose to assay the presence of viral genomes by PCR. This is an acceptable technique as long as the limitations are understood – it detects nucleic acids, not infectious virus.

Despite the presence of Zika virus RNA in seminal fluid for at least 60 days after infection, these mice are not likely to transmit virus after a few weeks. There is a lower limit of detection of the plaque assay – approximately 10 plaque forming units/ml – whether that would be sufficient to transmit infection is a good question.

Why Zika viral RNA and not infectious virus would persist for so long is an important and unanswered question that should definitely be studied.

Recently many papers have been published which demonstrate that Zika virus and Ebolavirus can persist in a variety of human fluids for extended periods of time. These results have been interpreted with alarm, both by scientists and by science writers. However, in most cases the assays were done by PCR, not by plaque assay, and therefore we do not know if infectious virus is present. Viral RNA would not constitute a threat to transmission, while infectious virus would.

The lesson from this study is very clear – in novel experimental or epidemiological  studies it is important to prove that any viral nucleic acid detected by PCR is actually infectious virus. Failing to do so clouds the conclusions of the study.

There are few excuses for failing to measure viral infectivity by plaque assays. Please don’t tell me it’s too much work – that’s a poor excuse on which to base selection of an assay. Even if your virus doesn’t form plaques there are alternatives for measuring infectious virus.

If you are wondering how a plaque assay is done, check out my short video below.

TWiV 428: Lyse globally, protect locally

The TWiVsters explain how superspreader bacteriophages release intact DNA from infected cells, and the role of astrocytes in protecting the cerebellum from virus infection.

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Bacteriophage superspreaders

bacteriophage modelBacteriophages are the most abundant biological entities on Earth. There are 1031 of them on the planet, and they infect 1023 to 1025 bacteria every second. That’s a lot of lysis, and it leads to the release of huge quantities of DNA that can be taken up by other organisms, leading to new traits. It seems that some bacteriophages are very, very good at releasing intact DNA, and they have been called superspreaders (link to paper).

In a very simple experiment, E. coli cells carrying a plasmid encoding ampicillin resistance were infected with the well studied phages T4 and T7 and also with a collection of 20 phages isolated from soil, water, and feces in Miami and Washington DC. After the cells lysed, DNA was extracted from the culture medium and introduced into antibiotic sensitive E. coli. Two phages, called SUSP1 and SUSP2, were thousands of times better at releasing plasmid DNA that readily conferred antibiotic resistance. These phages are superspreaders.

Superspreader phages can promote transformation by different plasmids, so their unique talent is not sequence specific. When these phages lyse cells, intact plasmid DNA is released. In contrast, phage T4 infection leads to degradation of plasmid DNA in the host cell. Superspreader phages lack genes encoding known  endonucleases – enzymes that degrade DNA, possibly explaining why plasmids are not degraded during infection. Other phages that lack such endonucleases, including mutants of lambda and T4, also promote plasmid mediated transformation.

Phages SUP1 and SUP2 don’t just spread plasmids to laboratory strains like E. coli. When crude mixtures of soil bacteria from Wyoming and Maryland were mixed with SUP1 and SUP2 lysates from E. coli, antibiotic resistance was readily transferred. One of the main recipients of plasmid DNA is a member of the Bacillus genus of soil bacteria, showing that superspreaders can move DNA into hosts of a species other than the one they can infect.

With so many bacteriophages on the planet, it is likely that there are many other superspreaders like SUP1 and SUP2 out there. The implication is that massive amounts of intact plasmid DNAs are being released every second. These DNAs can be readily taken up into other bacteria, leading to new phenotypes such as antibiotic resistance, altered host range, virulence, the ability to colonize new niches, and much more.

You might wonder if all that plasmid DNA, floating in the environment, can also enter eukaryotic cells – and the answer is yes. No wonder eukaryotes didn’t invent anything.

TWiV Special: Vincent Munster on MERS-coronavirus and Ebolavirus

At the Rocky Mountain Laboratory in Hamilton, Montana, Vincent speaks with Vincent Munster about the work of his laboratory on MERS-coronavirus and Ebolaviruses.

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TWiV 427: It was a DURC and UV light

The TWiVoids discuss the March for Science, the GOF moratorium, and a classic virology paper on mapping the gene order for vesicular stomatitis virus.

You can find TWiV #427 at microbe.tv/twiv, or listen below.

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