Polysomally Protected Viruses
PPolysomally Protected Viruses
Michael Wilkinson , , David Yllanes and Greg Huber Chan Zuckerberg Biohub, 499 Illinois Street, San Francisco, CA 94158, USA School of Mathematics and Statistics, The Open University,Walton Hall, MiltonKeynes, MK7 6AA, UKE-mail: [email protected]
January 2021
Abstract.
It is conceivable that an RNA virus could use a polysome, that is, a stringof ribosomes covering the RNA strand, to protect the genetic material from degradationinside a host cell. This paper discusses how such a virus might operate, and how itspresence might be detected by ribosome profiling. There are two possible forms forsuch a polysomally protected virus , depending upon whether just the forward strand orboth the forward and complementary strands can be encased by ribosomes (these willbe termed type 1 and type 2, respectively). It is argued that in the type 2 case theviral RNA would evolve an ambigrammatic property, whereby the viral genes are freeof stop codons in a reverse reading frame (with forward and reverse codons aligned).Recent observations of ribosome profiles of ambigrammatic narnavirus sequences areconsistent with our predictions for the type 2 case.
1. Introduction
A canonical model for the structure of a virus [1] consists of genetic material encasedin a capsid composed of a protein shell. A simpler model has also been observed,termed a narnavirus (this term is a contraction of ‘naked RNA virus’). The narnavirusexamples that have been characterised appear to be single genes, which code for an RNA-dependent RNA-polymerase (abbreviated as RdRp) [2]. It appears to be advantageousto the propagation of a virus if the genetic material can be encapsulated at some stagein its replication cycle, and it appears natural to ask whether some very simple virusescould co-opt part of the machinery of the host cell in order to build a container. Themost natural candidate is to make a covering out of ribosomes, which already containan internal channel that can bind to RNA. If viral RNA can be completely coveredwith a chain of ribosomes, it could be well protected from defence mechanisms of hostorganism, because the exterior of the package presents molecules which are part of thehost cells. This paper discusses how a class of very simple viruses could make a containerfor their genetic material out of ribosomes, resulting in a class of RNA viral systemswhich are, in some sense, intermediate between narnaviruses and conventional viruses.The covering structure, consisting of a chain of ribosomes attached to the viral RNA, a r X i v : . [ q - b i o . S C ] J a n olysomally Protected Viruses a ). The type of encapsulation that we propose is one wherethe ribosomes are stuck in position. This means that we must hypothesise a mechanismwhich creates the polysome shell by preventing ribosomes from detaching from the 3 (cid:48) end of the viral RNA (figure 1( b )), thus creating a ‘frozen’ polysome. We propose thatribosomes attach to the 5 (cid:48) end of the viral RNA chain and move along the RNA chainuntil they form a string of ribosomes which are in close contact, like a string of pearls(figure 1( c )).Cells have machinery to release ribosomes which are not functioning efficiently [9].In particular, ‘stalled’ ribosomes are released by a process known as ‘no-go decay’,abbreviated as NGD, which is an active field of study [10, 11, 12, 13]. The polysomallyprotected virus system would have to either disrupt the NGD process, or else infectcells where this process is defective. Given the complexity of the machinery required toimplement ‘no-go decay’, it must have many potential vulnerabilities.We can imagine two forms of this class of virus. In its simplest form, termedPolyProV1, a polysomal sheath is only able to cover the forward strand of the RNA.Creation of a complementary strand is a necessary part of the replication cycle of theviral RNA, and in the simplest form, the complementary strand is not protected. Wecan also propose that there exists a type of this viral system, denoted by PolyProV2,where both the forward and complementary strands can be protected by being encasedin a chain of ribosomes.We discuss what would be the characteristic properties of such a system, and howtheir presence might be detected. Both types, PolyProV1 and PolyProV2, may showdistinctive signatures under ‘ribosome profiling’ (see [14, 15] for a discussion of thistechnique), and we give an indication of what might be expected. We remark thatrecent experiments on a narnavirus system Culex narnavirus 1 , reported in [8] showprecisely the type of ribosome profile signatures that we describe (without explainingtheir form). We also argue that, in the case of PolyProV2 systems, there would be avery distinctive signature in the genetic code of the virus. The formation of a polysomewhich covers the whole of the strand requires that the RNA sequence should not haveany stop codons (that is, it should have an open reading frame, abbreviated as ORF).The genes of the PolyProV2 system would therefore have to have a reading frame which olysomally Protected Viruses Figure 1. (a)
A polysome consists of a number of ribosomes attached to a RNAmolecule (usually mRNA). The ribosomes attach to the 5 (cid:48) end and move along theRNA, translating polypeptide chains as they go. (b)
Our hypothetical polysomallyprotected virus is an RNA virus system including a gene that creates a ‘blocking’macromolecule (either a protein or possibly an RNA segment), which binds to arecognition site at the 3 (cid:48) end of the virus. Ribosomes are able to attach to the virusRNA at the 5 (cid:48) end, but are not released at the 3 (cid:48) end. (c)
The viral RNA becomescoated in ribosomes, which are frozen into fixed positions, and which form a protectivesheath. is devoid of stop codons on the complementary strand, as well as the forward strand. Wehave previously discussed the evolution of genetic sequences which are ‘ambigrammatic’,that is, readable in both forward and reverse directions, showing that stop codons inthe reverse-read direction can be eliminated even if the amino-acid sequence of a geneis strictly conserved [16]. We argue that a recent observation of two ambigrammaticsequences in the
Culex narnavirus 1 system reported in [17, 8] is a very strong candidateto be a PolyProV2 type virus.Ambigrammatic sequences have been observed in narnavirus systems, and it ispossible that the ORF on the complementary strand code for a functional protein.In a separate paper [18] we shall discuss criteria based upon statistical studies ofpolymorphism which could distinguish the PolyProV2 system from a narnavirus whichhas a functional gene on the complementary strand. Our results for
Culex narnavirus1 and for
Zheijiang mosquito virus 3 indicate that the complementary strands are not olysomally Protected Viruses stationary because their release at the 3 (cid:48) end is blocked.
2. Predicted properties
Let us assume that an RNA virus does use a ‘frozen’ polysome to create a capsule out ofribosomes, and consider what are the plausible consequences of this hypothesis. Thereare two questions that should be addressed. Firstly, how is the frozen polysome created?And secondly, is it possible to protect the complementary strand as well as the codingstrand of the virus?
The most natural hypothesis about the mechanism to create frozen polysomes is thatribosomes are prevented from detaching from the 3 (cid:48) end of the RNA. The simplestmechanism for this is for there to exist a macromolecule (a protein, or an RNA segment)which binds to the 3 (cid:48) end of the viral RNA to block ribosomes from detaching. At leastone gene would be required to code for this ‘end-stop’ macromolecule.The mechanism which freezes polysomes must have a quite specific switch, whichcan distinguish virus RNA from the host mRNA (if this were not the case, then the‘end-stop’ would inhibit all translation processes indiscriminately, damaging the hostcell). The required specificity would have to be achieved by a signalling sequence in thevirus RNA, such that the end-stop only binds when the signalling sequence is present.The only plausible location for the signal sequence is at the 3 (cid:48) end of the virus RNAchain, where the end-stop protein will bind.These considerations imply that the simplest polysomal virus would have two genes,one to make the RdRp to replicate the virus, and the other one to make a blockingmolecule to stop ribosomes from detaching from the 3 (cid:48) end of the viral RNA. In addition,there must be a recognition sequence at the 3 (cid:48) end of the virus chain. Eukaryotic cellshave mechanisms for releasing ‘stalled’ ribosomes [9, 10, 11, 12, 13], and if polysomallyprotected viruses exist, that may be associated with other virus genes which disrupt themechanisms which release stalled ribosomes.It is important to note that this picture implies a mechanism whereby the ribosomes olysomally Protected Viruses (cid:48) ends of viral RNA creating a reservoir of viral RNA which isprotected from degradation.
Replication of the virus RNA by the RdRp requires making a complementary copy. Inaddition to protecting the coding strand of the RNA, the polysomal virus could alsoevolve so that the complementary strand can be protected. Let us consider the additionalfeatures that are required to convert a PolyProV1 system, where just one strand isprotected, to a PolyProV2 system, where both the forward and the complementarystrands can be enclosed by a frozen polysome.For a typical RNA sequence there will be stop codons on the complementary strandwhich would cause ribosomes to detach, preventing the RNA sequence from becomingshielded inside a polysome. This can be avoided if the RNA sequence is ambigrammatic ,in the sense that it is readable, without encountering codons, in both a forward andreverse reading frame. Recently, it has been shown that it is always possible to create anambigrammatic sequence by substitution of codons by synonyms [16]. This mechanismgives a rapid route to evolving an ambigrammatic sequence, without detriment to thefunction of genes translated in the forward direction. The ORF for the complementarystrand must have the its codons aligned with the ORF for transcription on the forwardstrand [16].There is an additional requirement for the complementary strand to be protected:the 3 (cid:48) end of the complementary strand has to have a recognition sequence to signalthe end-stop protein to attach itself. This implies that there is a reverse complementof a valid recognition sequence at the 5 (cid:48) end of the coding strand. The simplestimplementation of this is if the 5 (cid:48) end has a sequence which is the reverse complementof the recognition segment at the 3 (cid:48) end of the coding strand.In this context, we remark that narnaviruses typically have a sequence CCCC at the3 (cid:48) end, and a complementary sequence GGGG at the 5 (cid:48) end. These sequences have beenshown to be important for the propagation of narnaviruses, [25, 26], but the mechanismwhich makes these termination sequences important has not been clear.Finally, consider the evolution of a PolyProV2 system from a PolyProV1 virus.The reverse-complement recognition sequence would have to exist on the 5 (cid:48) end as apre-requisite, but once this is in place, a partially ambigrammatic sequence can confer apartial advantage, so that the ambigrammatic property can evolve incrementally. Thereis no requirement for the reverse-read sequence to code for a functional protein. Beyondthe requirement that there are no stop codons, there need not be any selective pressureson the reverse-read sequence. olysomally Protected Viruses
3. Identification of PolyProV virus systems
Next we consider the general principles which could be used to provide evidence for theexistence of a PolyProV virus system. There are two approaches which could be used.Because the defining feature of PolyProV viruses depends upon their interactionwith ribosomes, ribosome profiling techniques should be important. In particular, weshould address how these would distinguish ribosomes which have become ‘frozen’ fromthose where translation is progressing. This approach could detect both PolyProV1 andPolyProV2 systems.The other approach is to use evidence from sequencing the viral RNA. The existenceof ambigrammatic genes would be an indicator of a PolyProV2 system. In this case weneed to consider how to distinguish signatures of a PolyProV2 virus system from otherpossible explanations of ambigrammatic sequences.
Ribosome profiling techniques [27, 28] are based upon mechanical disruption of polysomecomplexes formed by ribosomes and RNA, followed by RNA sequencing. The mechanicaldisruption creates RNA fragments which represent sections of the RNA strand whichwere covered by ribosomes moment when the polysome was disrupted, as shownschematically in figure 2( a ) and ( b ). The segments which lie under the ‘shadow’of a ribosome are amplified and sequenced. These segments are sufficiently long(approximately 35 nt) that their position in the genome can be (in almost all cases)uniquely determined. Ribosome profiling data is often illustrated by plotting thefrequency for counting fragments containing a base x as a function of the position ofthe base on the RNA chain. Higher counts are expected in regions where the ribosomesmove more slowly, and a typical ribosome profile plot has an appearance similar to thesketch in figure 3( a ).Consider how this technique can reveal jammed ribosomes, as indicated in figure1(c). In order to understand the form of the expected profile, it is necessary to appreciatethat the measured profile is a product of two factors: the desired signal, which isthe ribosome coverage over a given nucleotide, must be multiplied by a factor whichrepresents the amplification number of the RNA fragments. The latter is related to thefragment sequence in a manner which is deterministic, but where the actual relationshipis unknown. For this reason, the polymerisation amplification factor of a segment mustbe regarded as a random variable.There is, however, one simple observation that we can make about the amplificationnumber. Because amplification involves successive replications of both the segment andits reverse complement, the amplification factor of the reverse complement of a segmentmust be highly correlated with that of the segment itself.Now consider the ribosome profile resulting from stalled ribosomes. According tothe PolyProV model, ribosomes will be prevented from detaching from the 3 (cid:48) end, andwill form a close-packed array along the viral RNA, resembling a string of pearls. Our olysomally Protected Viruses c ). In particular, all of the bases under the shadow of a stalledribosome are represented by the same population of RNA fragments, which have thesame amplification factor. As we move along the chain, we encounter nucleotides whichare under the shadow of an adjacent ribosome, and which are represented by a differentRNA fragment, with a different amplification factor. At this point, the sequences whichare being PCR amplified change abruptly. The replication rate of the new sequence islikely to be different, so that the heights of the plateaus will be different, forming anapparently random sequence, as illustrated in figure 3( b ). The plateaus all have thesame width, approximately 35 nt. This is in contrast to the results of ribosome profilingfrom an mRNA molecule which is being translated, where the there are many differentfragments containing a given base.Our discussion of the ribosome profile of a PolyProV virus assumes that most ofthe ribosomes which are attached to viral RNA is stalled. It is possible that somesmall fraction of the viral RNA is still being translated by moving ribosomes, while the‘plateau’ profile is visible. The plateau structure is also compatible with most of theviral RNA in a cell not being bound to ribosomes.The separation of the frozen ribosomes may be subject to variations, becausedifferent base sequences bind to the ribosomes in a slightly different configuration. It isalso possible that the length of the RNA strand which is covered by a ribosome mightfluctuate as a function of time. These fluctuations would accumulate as we move furtherfrom the 3 (cid:48) end. In this case the plateaus in the ribosome profile plot would become lessdistinct as we move further from the 3 (cid:48) end, as illustrated in figure 3( c ). The extentto which the ribosome profile plots would resemble figure 3( c ) rather than figure 3( b )would have to be determined by experiment, but it would be expected to be a consistentfeature of PolyProV systems.In the case of a PolyProV2 system, the ribosome profile plot for the reverse strandswould also show a sequence of plateaus, which would be most distinct at the 5 (cid:48) end ofthe chain. If the plateaus in the profiles of both forward and complementary chainsoverlap, they could be ‘in phase’, or ‘out of phase’, or somewhere in between. Forexample, if the length of the region occupied by a ribosome is dependent on the sequenceof bases that the ribosome covers, there will be apparently random variations in thelengths of the plateaus illustrated in figure 3( b ), and the plateaus for the forward andcomplementary strands will be in phase over part of their length, and out of phase inother regions, as shown in figure 4. When the ribosome shadows of the forward andreverse strands are in phase, the segments which are amplified by the PCR process arecomplements of each other. When the plateaus are in phase, we expect the heights of olysomally Protected Viruses Figure 2. ( a ) In ribosome profiling experiments, polysomes are disrupted and theRNA fragments which are under the ‘shadow’ of a ribosome (approximately 35 ntlong) are polymerised and sequenced. ( b ) If the ribosomes are moving along thepolysome, the fragments containing a given base (indicated by x ) will have that basepositioned at any point within the fragment. Some of these fragments contain anotherbase y , whereas others do not. ( c ) If the polysomes are jammed, all of the fragmentscontaining a given base will be very similar in structure, and have the base x locatedat approximately the same position within the fragment. In this case the fragmentsthat contain base x always contain base y , but base z is always found on a differentfragment. the plateaus for the forward and complementary chains to be significantly correlated,because the polymerisation reaction involves multiple replications of both forward and complementary images of the fragments. In the regions where the ribosome shadowsare out-of-phase, as in the centre section of the strand shown schematically in figure 4,the plateau heights of ribosome profiles from the forward and reverse strands will beuncorrelated. We have proposed that a PolyProV2 system could be detected by finding ambigrammaticviral genes in sequencing studies. The detection of ambigrammatic sequences is anunambiguous signal, and it is one which has already been observed in RNA virussequences. It is necessary, however, to consider whether alternative explanations are olysomally Protected Viruses position5′ 3′5′ approx35nt (b)(c)(a) Figure 3. ( a ) Illustrates the appearance of a typical ribosome profiling plot, observedwhen ribosomes are moving (right to left) along the RNA. ( b ) If the ribosomes arefrozen in fixed positions, the plot will show a sequence of plateaus. The width of eachplateau is the length of the shadow of a ribosome, approximately 35 nt. ( c ) If theseparation of the jammed ribosomes fluctuates randomly as a function of time, theplateaus may become less distinct as we move away from the 3 (cid:48) end. viable.The possible explanations for observation of an ambigrammatic viral RNA sequencefall into two classes. It might be that the reverse-readable sequences are expressed asproteins, which serve some function in facilitating the propagation of the virus, forexample, the protein might poison defence mechanisms of the host cell, or it might forma complex with the viral RNA which provides some protection. The other possibility isthat the ambigrammatic property provides some other advantage, without necessarilybeing expressed as a protein. The lack of stop codons facilitates the association ofribosomes with the complementary RNA strand, so any plausible mechanism wouldhave to involve ribosomes in some way.There are three lines of evidence which can help to decide on the mechanism. Thetheory of the PolyProV2 system is consistent with the evolution of the complementarystrand sequence being neutral, because there is no role for the amino-acid sequence codedon the complementary chain (although some of the protein may be translated). One testof whether a sequence codes for a protein is to look at the ratio of non-synonymous tosynonymous mutations which will be denoted by R = ∆ N/ ∆ S , (where ∆ N and ∆ S are, olysomally Protected Viruses Figure 4.
In the case where both forward and complementary strands of a PolyProV2system are detected in ribosome profiling, the plateaus on the complementary strandsmay overlap. If the lengths of the plateaus have some dependence upon the basesequence which is covered by the ribosome, the plateau widths vary apparentlyrandomly, so that the forward and complementary strand plateaus are ‘in-phase’ insome regions, and ‘out of phase’ in others. The plateau heights are correlated whenthe strands are in phase. respectively, the number of non-synonymous and synonymous mutations). We expect R to be small when a readable base sequence is a functional gene coding for a protein, andthe R value for the forward sequence which codes for the RdRp is very small, indicatingthat this gene is strongly conserved. If both the forward and the complementary strandscode for a protein, we might expect mutations which are synonymous for both forwardand reverse transcription would be better tolerated. We shall report in detail uponan investigation of this approach elsewhere [18]. For both the Culex narnavirus 1 and
Zheijiang mosquito virus 3 , the evidence indicates the the complemenary strands ofknown ambigrammatic virus segments do not code for functional proteins.Ambigrammatic sequences have been observed in a variety of simple RNA virusgenomes [16], but they are undoubtedly a rare phenomenon. Given that ambigrammaticsequences are rare, if two or more genes within a virus infection system are found tobe ambigrammatic, this would be very unlikely to be the result of two functional genesarising on the complementary strand. An observation of the simultaneous detection oftwo or more ambigrammatic genes would strongly favour models, such as the PolyProV2 olysomally Protected Viruses
Recently, a mosquito-hosted narnavirus system (
Culex narnavirus 1 ) has been found tobe associated with two ambigrammatic genes [17, 8]. It has properties which make it astrong candidate to be a polysomal virus (see [17, 8] for a discussion of the experimentalevidence):(i) There is a viral RNA segment which codes the for the RdRp, and which resemblesa narnavirus, but which has the property of being ambigrammatic, with forwardand reverse codons aligned.(ii) Infection with this sequence is strongly associated with the presence of anotherRNA sequence, which was referred to in [17] as the ‘Robin’ sequence.(iii) The Robin sequence is also ambigrammatic over its entire length (about 850 nt),with the codons of the forward and reverse ORFs aligned. Neither forward norreverse directions are homologous to known sequences.(iv) Ribosome profiling experiments show a ‘plateau’ structure [8], which closelyresembles that which is sketched in figure 3( b ). The plateaus are seen in ribosomeprofiles of both the RdRp gene and the Robin sequence. There is no evident lossof definition of the plateaus on moving away from the 3 (cid:48) end, as illustrated infigure 3(c). This indicates that the packing of the ribosomes is very tight.(v) The ribosome profile experiments detect the complementary strand of both theRdRp and the Robin sequence. Both of the complementary strands have ribosomeprofiles with plateaus.(vi) When the ribosome profiles of the forward and complementary strands arecompared, the heights of the plateaus are correlated when they are in phase witheach other, as illustrated in figure 4.(vii) The companion and RdRp coding sequence share the feature of havingcomplementary terminal sequences: both the RdRp and companion segments haveone end terminating with CCCC, while the opposite end terminates GGGG.These features are consistent with the properties of a PolyProV2 type virus system,as described above. In particular the fact that the two sequences are strongly correlatedstrongly implies that both are required for a viable infection. There is no evidence olysomally Protected Viruses
4. Discussion
We have proposed that viral RNA can be protected from degradation inside polysomesif these are ‘frozen’. This hypothesis explains recent observations [8] of distinctiveribosome profiles of some narnaviruses. It also explains the existence of ambigrammaticsequences, because both phases of replication of an ambigrammatic gene can beprotected. The use of protective polysome coverings may prove to be a widely distributedproperty of viral systems.
Author Contributions
MW produced a draft of the manuscript following discussions with the other authorsabout the recent discovery of a narnavirus system which has two ambigrammatic genes.All authors contributed to writing the manuscript, and reviewed the manuscript beforesubmission.
Acknowledgments
We thank Hanna Retallack and Joe DeRisi for discussions of their experimental studiesof narnaviruses. GH and DY were supported by the Chan Zuckerberg Biohub; MWthanks the Chan Zuckerberg Biohub for its hospitality.
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