Viral Replication of RNA Viruses & Antiviral Strategies

Joanna Fernandez Fuentes

Department of Chemistry

San Jose State University

November 4, 2021

Peer Review by Huy Dang, 015185820

Nov 30, 2021

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Table of Contents

I. Abstract ……………………………………………………………………………………

II. Introduction ……………………………………………………………………………….

III. Viruses during Evolution ……………………………………………………………….

IV. Role of tRNAs in Viruses………………………………………………………...………

V. Other Factors that Play a Role in RNA Viruses……………………………………….

VI. Antiviral Strategies……………………………………………………………………….

VII. Covid-19……………………………………………………………………………….

VIII. Conclusion……………………………………………………………………………….

IX. References ……………………………………………………………………………….

X. Figure Legends ………………………………………………………………………….

XI. Figures and Tables ……………………………………………………………………

Include page number would be helpful.

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Abstract

With the recent health crisis the world experienced and is still experiencing with

Covid-19, questions regarding RNA viruses came to light. RNA viruses contain RNA

and, in some cases, may contain double-stranded DNA as well. To study these viruses,

their detection is required. Immunofluorescence analysis uses antibodies that can bind

to the desired protein through a process known as staining. Because double-stranded

RNA triggers a natural immune response, it is easily detected by immunofluorescence

staining. It was not expected that negative-strand RNA viruses would be detected in

staining but was not the case with four different types of viruses. Most RNA viruses

were found in the cytoplasm, but a new finding determined that some viruses could also

be stained and detected in the nucleus. Further research shall be done to determine

how different types of RNA, most specifically tRNA, play a role in gene expression to

understand and develop antiviral strategies.

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Introduction

Although not all living systems are made or function in the same way, these

systems acquire, store, and use important coding information that makes them unique.

One thing that all these living systems have in common is the composition of cells, or

the structural unit of living matter making up the organisms and tissues. Each organism

has a different number of cells, but to say the least, they all have many cells. In fact, the

human body has an estimated amount of thirty trillion cells. Inside the cells, there are

smaller components that make it up. These three main components of a cell are the cell

membrane, the cytoplasm, and the nucleus. The cell membrane serves a selective filter,

controlling what can go in and out of the cell. The cytoplasm is the fluid inside the cell

that helps proteins be formed and other chemical reactions to occur. Most importantly,

the nucleus is the heart of the cell. In here, information that can be retrieved or

duplicated in genetics is stored. This information is what is known as deoxyribonucleic

acid.

Deoxyribonucleic acid, most commonly known as DNA, is a double-stranded

helix chains of polynucleotides that carry coding information that is used in protein

synthesis. DNA works hand-in-hand with RNA, or ribonucleic acid. Besides the fact that

RNA is single stranded compared to DNA, RNA has a sugar ribose, while DNA contains

the slightly different sugar deoxyribose. The nucleotides within these molecules are also

fairly similar, including adenine, guanine, and cytosine. The exception to these lists of

nucleotides is that DNA contains thymine. On the contrary, RNA contains of uracil

instead. This difference in nucleotides allows thymine to substitute uracil and

deoxyribose for ribose when translating from RNA to DNA. It is assumed that RNA had

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to come before DNA because DNA could have never been possible with the existence

of RNA. These polynucleotides represent endless combinations of coding in which the

Last Universal Common Ancestor (LUCA) describes that a living system had to have

evolved from possible combinations that now only represent their original combination in

smaller ways (Subramanian et al., 2020).

Viruses are considered living systems, meaning they contain the properties

mentioned above, including DNA or RNA. A DNA virus has a genome made of

deoxyribonucleic acid that can be replicated. Meanwhile, an RNA virus contains RNA as

its genetic material. RNA viruses are considered more prone to mutations, meaning

they can be more be more dangerous and cause more harm due to its instability in

nature. It is assumed that the exposure of RNA viruses occurred in the interbreeding

between Neanderthals and modern humans. This exposure could have been either by

direct contact or through their environment that was once shared between the two

groups of individuals. Thus, alleles that fought the viruses could have been passed

along as well if there were such thing at the time. These alleles can be seen as

antibodies. Up until today, there is very limited information that can lead to antiviral

strategies to halt the mutations occurring in these viruses to keep them from expanding

even more (Enard et al., 2018). This is very terrifying because an RNA virus causes

severe illness such as fevers and respiratory complications that are not able to be

treated once the virus is in the human body (Leyssen et al., 2008). To attempt to solve

this issue, the detection of these viruses in living organisms is first required. An initial

host immune response to the presence of RNA, and DNA viruses as well, is the

production of double-stranded RNA. Immunofluorescence analysis is a process in which

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staining of a desired protein occurs using antibodies. This staining differentiates the

desired proteins from other proteins with a color change that is easily seen. The location

in which these strands of RNA are found can also be useful for understanding the virus

itself in a better way (Son et al., 2015).

There are many types of RNA that occur within living organisms, but there will be

several that will be emphasized more during this text. To synthesize proteins and

reproduce themselves, viruses rely heavily on transfer RNA, also known as tRNA. tRNA

links messenger RNA and the amino acid sequence through the ribosome. Messenger

RNA is called mRNA. This allows for the coding information to carry on and arrive at its

desired destination. The host tRNA’s coding information is tweaked at times by the own

virus for its own advantage. A prime example of this is tRNA-derived fragments. tRFs

stands for tRNA-derived fragments. tRFs result from enzymatic cleavage, making the

viral replication more assessable and easier to happen. tRNA often changes genetic

codes by modification of coding or demethylating adenosine in mRNA (Nunes et al.,

2020). The modification that occurs in enzymes can be described as RNA writers or, on

the other hand, the demethylation as the RNA erasers. Therefore, in addition to the

detection of the RNA viruses, the detection of tRNA is crucial to understand the role it

plays in the replication of the virus. Instead of using staining as a method of detection,

nucleic acid isotope labeling mass spectrometry (NAIL-MS) is performed to detect this

transfer of RNA coding. NAIL-MS dives deep into the mechanism that occurs within the

RNA to determine what amino acids sequences have been made and modified

throughout the process. This means that there should be a comparison between the

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initial pre-existing tRNA pool and the synthesized tRNA pool of new coding information

(Heiss et al., 2017).

The reproduction of RNA viruses can happen in many ways, but for growth, there

are only two requirements for it to occur. One of them demands that the growth occurs

through the replication of their RNA genomes and the other is by synthesizing their

nucleic acid and proteins. This is what is known as their genetic factors influencing the

growth. However, environmental factors can also affect the growth of the virus. This

concept itself is very limited with information, but there is some basic knowledge that

has been gathered throughout the years. Most viruses are found in heterogeneous

locations within the living system. This allows them to adapt to different environments

very easily and settled. As their environment keeps changing around them, nucleic acid

adaptation and rearrangement occur that gives them just enough adaptation to survive.

In other words, the viruses maintain their functional and structural elements, only

making them more adaptable to its environment (Ferron et al., 2021). Considering this

information, it is assumed that this can give RNA viruses an advantage in the presence

of an antiviral drug, making it very hard to inhibit. As they expand, enzymes are required

to catalyze the reactions occurring. These mutations rates will vary anywhere from 10-6

to 10-4 substitutions per nucleotide. These mutations rates are inversely proportional to

the genome size. This means the bigger the genome, the slower the mutation rate will

be and vice versa (Bradwell et al., 2013). This can be expected as these genomes will

require more mutations to occur in order to change the function of the virus itself (Zhu et

al., 2009). In past studies, there has been a positive correlation between mutation rates

and the virus's ability to cause damage to a host cell. This allows for the assumption

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that high rates of mutation increase the adaptability of the virus itself. As mutations

occurs, errors per nucleotide may occur. This questions whether the virus prefers it

have quick mutation rates or correct mutations that lead to fidelity (Fitzsimmons et al.,

2018).

As expected, today drugs that are commonly used to treat RNA viruses, such as

the Covid-19 vaccine, are inhibitors that bind to an enzyme and prevents a catalysis of

reaction to occur by attacking the active sites. These antivirals are very limited and the

production of them is necessary to ensure the safety of individuals and avoid pandemics

from arising. Very small amounts of these drugs are on the market, making it also very

difficult to understand and attempt to get more information. Due to the fact that some

RNA viruses have broad-spectrum inhibition, some RNA viruses can be dealt with

similarly. For example, severe acute respiratory syndrome infections (SARS) were

believed to be treated with ribavirin (Barnard et al., 2006). That unfortunately may not

always be the case. Considering all RNA viruses have coding that makes them unique

in their own ways, not all attempts will work for all RNA viruses. Instead, different

strategies will have to be made to suit different viruses. Another important point to

consider is in what organism is the RNA virus in. Both plant and humans suppress RNA

silencing through their mechanism (Andersson et al., 2005). However, vertebrates

induce the synthesis of proteins that have a general inhibitory effect on virus

multiplication. Studying the genomics of enzymes that occur within the replication

process can be a pivotal point for developing antiviral drugs against these viruses

(Leyssen et al., 2008).

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 In this paper, the function of RNA viruses will be examined to understand the

mechanisms involved in the growth of the virus. This can potentially lead experts to

develop antiviral strategies against most, if not, all RNA viruses.

Viruses in Evolution

For evolution to occur, organisms’ coding information mutates constantly,

including the process of transforming RNA into DNA by substituting thymine for uracil

and deoxyribose for ribose caused by nucleobases of probiotic chemistry. The

polynucleotides also go through both physicochemical and biophysical properties that

allow for many functions such as encoding to transition of information to even

orientation of strands to occur. It is considered that the properties of both DNA and RNA

have specific mutations of their nucleotides that are predetermined for their own benefit

of reproduction. In other words, mutations do not occur by chance, but as the

organism’s mechanism for adaption. By exerting both RNA and DNA molecules under

special conditions that are similar to primordial earth, these molecules were studied to

understand their evolution. Considering that double-stranded RNA is only present in

viruses, it is assumed that it was able to store coding information for the first replicators.

The lack of double stranded RNA in prokaryotic and eukaryotic organism after time can

then propose the idea that DNA took over the role of storing information and RNA was

left to translate that coding information into amino acids (Subramaniam et al., 2020).

When thinking about humans, it is particularly impossible to see humans as

anything other than what we are accustomed to today. With great historians, it was

brough to our attention that there were now-extinct humans on the Earth way before we

existed. These archaic humans are now referred to as Neanderthals about forty

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thousand years. Their extinction is still being questioned, but heavy evidence points to

the climate change, disease, and assimilation into what we consider modern genome as

the cause for their disappearance. Despite this, it is believed that the modern human

was around when Neanderthals were evolving, and they indeed did interbreed in two

occasions (Enard et al., 2018). Today, it appears that natural selection has removed

most alleles from the modern human population. If an allele is found, it is considered to

be very weak. (Juric et al., 2016).

In the first occasion that occurred eight hundred thousand years ago, there was

no trace left if Neanderthal genomes in modern humans, but much rather modern

genomes in modern human genomes were found in Neanderthals. This could explain

their evolution as their genomes were overpowered by the genomes found in modern

humans. However, during the second interbreeding, there were more segments of

Neanderthal genomes found in modern humans, more specifically, in non-African

modern humans (Figure 2).With this interbreeding that occurred, it is possible that

these living organisms exchanged viruses at the time, caused by a direct contact or via

their environment that was shared within the two (Enard et al., 2018). These viruses are

called VIPs, or virus-interacting proteins. These proteins can also be viral DNA or viral

RNA. These interactions can be caused by the hijacking of host transcription machinery

by viral proteins or the the binding of viral coat proteins to host membrane.

When proteins in organism interact with VIPs, they tend to adapt at much higher

rates than those proteins that do not interaction with viruses. In fact, using the

McDonald-Kreitman test (MK test), VIPs drive an estimated amount of thirty percent of

all the protein adaptation came in humans alone (Enard et al., 2016). This may indicate

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that viruses play a big role in the evolutionary change that occurs within organisms.

CRF, or statistic’s method of Conditional Random Field, was applied to study the

patterns of segments between the Neanderthals and modern humans. It was then

concluding that the DNA of Neanderthals contained coding that allowed for adaptation

of RNA viruses. Similar to the idea that interbreed of viruses could have been passed

on from the Neanderthals to the modern humans, these adaptations against RNA

viruses could have also been given by the Neanderthals during the exchange (Enard et

al., 2018).

Role of tRNA in RNA Viruses

Viruses belong to the large variety of pathogen groups that are not too simple to

understand because there are just so many of them. This can pose a threat to the

global disease control due to their unknowns and their lack of treatment against them.

These viruses differ from one another in many ways, including their structure, replication

process, and gene expression. In a case of an outbreak, this can come with heavy

economic and mortality costs in the hands of the public. These viruses are broken down

into seven different classes with varying replication and gene-expression strategies

(Figure 1). Within these seven classes, a large number of viruses fall under RNA

classified viruses, including Ebola, Hepatitis C, and SARS (severe acute respiratory

syndrome). These viruses are then broken up into subcategories: (+) RNA, dsRNA, and

(-) RNA (Ahlquist 2006). RNA viruses are considered more dangerous than the other

DNA viruses and reverse-transcribing viruses due to the fact that RNA viruses have

very high mutation rates compared to DNA viruses. These mutation rates often make it

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hard for vaccines to be available considering they are always changing their genetic

coding, making it more difficult to target desired proteins.

When thinking of mutations, most are often assumed to be dangerous with

harmful consequences because of their association to mistakes during cell division in

DNA. However, that may not always be the case, especially when discussing RNA

viruses. On the contrary to this belief, high rates of mutations tend to favor the viruses

because it led to more adaptability within the living organism (Fitzsimmons et al., 2018).

It is also good to keep in mind that RNA is just as likely to go through mutations as DNA

is. As a matter of fact, there are many reasons as to why mutations occur including

exposure to ionizing radiation or chemicals known as mutagens, or infection by already

present viruses within the organism. Mutations rely heavily on RNA to reproduce,

especially on tRNA as its role to match amino acids to the respective codons in mRNA.

The process in which tRNA sequencing occurs have evolved through time due to

its structure, yet it serves the same purpose. Similarly, as to how mutations occur for the

purpose of reproduction, RNA viruses modify coding within the host tRNA for their own

good. This process is done by selecting host tRNAs from different pools found within the

organism to lead to their desired viral protein translation. This manipulation can also

occur once the transcriptional modification has occurred too. This is what is known to be

the post-transcriptional modification. This is often caused by infected cells with different

RNA viruses (Nunes et al., 2020). Although these modifications appear to only benefit

the virus itself, understanding how they work with the help of tRNA is crucial to creating

antiviral strategies to prevent the modification from occurring in the first place. In hopes

of control the expression of tRNA, pre-existing pools and synthesized tRNA

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modifications are required to understand their mechanism. This creates a comparison

between two isotopes using NAIL-MS. (Heiss et al., 2017). It is determined that tRNA

modifications are not always constant. Their modification increases and decrease

during the growth of the protein.

As important as the role of tRNA is to the development of viruses and its

reproduction, tRNA is something that should be taken very carefully into account due to

the posed risks it carries. Besides allowing RNA viruses to reproduce and get stronger,

in the past, tRNA has been associated with misregulation to cancer. tRNA MET
i is an

initiator in the process of cancer and catalyzes the reaction below?

ATP + L-methionine + tRNAMet ⇌ AMP + diphosphate + L-methionyl-tRNAMet

MET
Studying the effects of overexpression of tRNA i was a difficult challenge because this

changed other levels of tRNA found in the organism. This drastic change of the

expression profile increased the cell metabolic activity. This finding suggests that

cellular regulation when it comes down to tRNA expression is much more complex and

difficult to understand. Therefore, it should not be underestimated. (Pavon-Eternod et al,

2013).

Other Factors that Play a Role in RNA Viruses

Despite the heavy load of a role that tRNA plays in RNA viruses, there are other

factors that contribute to the maintenance of RNA viruses and their consequences. As

mentioned before, the process of encoding sequences was not random due to the

biochemistry and biophysical properties involved, the crossroads of RNA genomes do

not happen by chance either. In fact, genomes expression started with proviruses, also

known as replicators, that resembled RNA viruses very close. These proviruses have

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expanded their sizes like the RNA ancestors. Eventually, it is believed those proviruses

evolved their own polymerase while RNA viruses evolved through their hosts. This

expansion in size is related to the lack of dependency of polymerase sequencing. In

order for expansion to also occur in RNA viruses, methyltransferases (MTase) should

be present (Ferron et al. 2021).

Another factor that plays a role in the maintenance of RNA viruses is what type of

RNA makes up the virus itself. Many RNA viruses are double-stranded RNA viruses.

This means that the RNA that makes up the protein is a shape like a double helix,

winding over one another over and over. Some other viruses are limited to one a single-

stranded RNA within its complex. On the contrary to the double helix RNA, as given by

the name itself, a single-helix RNA is only having one strand of RNA. A single stranded

RNA is able to fold into more shapes rather than the double stranded RNA virus it has

more ability to move. On the contrary, the double stranded RNA viruses are more

limited to its movement due to its stiffness. This limited movement prepares for the

induction of host immune response.

Understanding the differences between single and double stranded RNA can

help play a role in the antiviral strategies taken to assure that they contain overpower

the organism that was infected. Therefore, it is crucial to know which stranded RNA the

virus is made up of. With this in mind, a new analytical technique was adopted in the

process. It is called Immunofluorescence Analysis, where a desired or targeted protein

is fluorescence. Son and colleges performed this experiment using both infections of

double stranded and single stranded RNA viruses. Expecting only to receive a

florescence indication of the presence of a double stranded RNA, they were shocked to

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see that some single-stranded RNA was also detected using this method (Son et al.,

2015). This can discovery suggests that even more viruses can be detected than priorly

acknowledge, leading to some sort of knowledge being learned from them.

Besides the component insides the actual virus, the environment in which the

virus is surround can affect how the virus is structured. To be more specific, the

environment can affect the size of the RNA virus. Their requirement to provide growth

within their protein is to replicate using their own polymerase and biosynthesizing using

their host cells. However, it may be difficult to understand how exactly these

environments found within the living organism affect the size of the virus directly

because the particles often interact with different host cells. Zhu and colleges used

isolated BHK (baby hamster kidney) cells that were infected with a virus. To ensure that

the virus injected would reproduce within the cell, more than one type of virus was

injected. This injected occurred at different phases of the cell development.

Observations were taken into account, and it was determined that there was no set

pattern as to how many virous particles were produced once infected with a virus. The

amount of these progeny particles ranged from fifty to eight thousand Zhu et al., 2009).

This suggest that there is a board fitness distribution that occurs when cells are injected

with some sort of virus (Figure 3). This is something to consider because this can

potentially mean that an RNA virus can inhibit itself in a cell at any point of their growth

development.

As mentioned before, there are many types of RNA viruses that are known of.

There is also a big possibility that there are more unknown RNA viruses that are yet to

be discovered. Due to their fast mutations ranging from 10−6 to 10−4substitutions per

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nucleotide, it is hard to keep up with their mechanisms in living organisms. It was

believed that these mutation rates varied differently depending on their genome size in

an inverse way. For example, as genome size increases, their mutation rate decreases.

Using bacteriophage GB, one of the smallest known ribovirus in size, the mutation rate

was measured at 1.4 x 10−4 substitutions per nucleotide (Bradwell et al., 2013). This

reinforces that inversely correlated relation between mutation rate and genome size.

This further suggests that viruses with smaller may be better equipped to adapt better

than viruses with larger genome size.

Antiviral Strategies

Most accounted for viruses tend to be RNA viruses which carry big risks such as

respiratory diseases and high fatality rates. Having drugs that can inhibit this virus are

crucial for the evolution for mankind, especially when the virus is transmitted from an

animal reservoir (Bray 2008). Their complexity makes it difficult to have appropriate

antiviral against them because they mutate so quickly, and their mechanism varies

widely. This mechanism difference starts from the entry and uptake of the virus itself.

For example, a picornavirus binds to a host cell receptor in the fold of the capsid. This

causes the protein’s rigidity to increase and therefore, the uncoating of the cell cannot

occur. Coronavirus, on the other hand, attach to the desired protein as a spike protein.

These spike proteins carry carbohydrates in which the organism’s immune system

detects as foreign or unwanted (Leyseen et al., 2008).

A common antiviral strategy against RNA viruses are cellular targets with

different functions. In some cases, the host cell metabolism is changed, preventing the

reproduction of the viruses. This interference of the building blocks within the cell can

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either slow down the production of the virus particles or even stop them completely. The

downside to this method is that host cells will also provide a cytostatic effect, where

selectivity will not occur (Leyseen et al., 2008).

In other cases, the interaction of the virus and the host cell and virus can be

avoided at all costs using inhibitors. These inhibitors block the virus from interacting with

the active site of the protein, causing the production of these viral particles in the

organism. As ideal as this sounds, the idea of complexity within RNA viruses is

reintroduced because all RNA viruses are different and will require different inhibitors to

prevent the interaction of the virus to the desired protein (Leyseen et al., 2008). A

common inhibitor is T-705 against influenza A virus. Its mechanism is completed by

inhibiting influenza virus RNA polymerase through competition with GTP (Figure 4). An

antiviral such as this one can save the lives of the twenty percent of individuals who get

influenza A virus here in the United States (Spurgers et al., 2007). To focus more on

today’s issue, there will be a strong emphasis on SARS-CoV, mostly known as Covid-

2019.

Covid-19

Coronaviruses are one of the very few and most recent viruses that have been

experienced the generations alive during the twenty first century. These viruses are so

adaptive to an organism’s environment that they can cause respiratory and intestinal

infections in both humans and animals. They were not feared until the outbreak of

SARS, the severe acute respiratory syndrome, in 2002 that originated from Guangdong,

China. This caused very mild flu symptoms. A decade later, another coronavirus

appeared in the Middle Eastern, giving it its name of MERS- CoV, or Middle East

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respiratory syndrome coronavirus. Both of these viruses were assumed to have started

in bats and then the passed into humans. (Cui et al., 2019). Today, there is another

coronavirus that haunts the human population once again.

With the name of Covid-19 or SARS-CoV-2., this virus caused a lot of fear and

brough uncertainty to the world that was once described as normal. It all started when in

December of 2019, Chinese patients arrived at the emergency room with symptoms

related to pneumonia. All these patients had one thing in common: they had all been to

a seafood market in Wuhan, China (Zhu et al., 2020). It was evident that a virus had

spread out to the public and contaminated all individuals present at the same location. It

did not take long for this virus to spread throughout the rest of China, forcing restrictions

to be put into place. Just like a wildfire, soon the rest of the world began to experience

the same dilemma that China faced.

Given by the name, there is a close relationship with between SARS coronavirus

and Covid-19 in terms of its genetics in which the virus attacked the virus report in the

lungs. This is what causes shortness of breath and other respiratory complications. In

efforts to understand covid-19 better, a group of patients were studied at the end of

January 2020 in Munich. These patients were middle-aged adulted with no underlying

health conditions. Patients were tested for Covid-19 using the RT–PCR from

nasopharyngeal swab specimens during their hospital visit starting with the day they got

their first symptom. All swabs starting from day one to day five of their symptoms

resulted in a positive test for Covid-19. There was an average RNA load of 6.76 x 10 5

copies per swab taken. In comparison to SARS, it would tend to take about seven to ten

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days for an RNA load of 5 x 105 copies per swab to be obtained. It was impressive to see

this value was higher in less than five days. Beyond the five days, the patients test

resulted varied. The longest period in which an individual tested positive for Covid-19

was twenty-eight days (Wölfel et al., 2020).

After Covid-19 was declared as a pandemic, a mortality fear in mind after the

eight thousand cases the resulted in seven hundred and seventy deaths back in 2002,

motivated experts to start their work towards an antiviral that could potentially stop the

growing virus (Burrer et al., 2007). Clinical trials were being held all over the world in

attempts to develop a vaccine. During one specific trail, BNT162b1 was formulated.

BNT162b1 is a nucleoside-modified mRNA vaccine that encodes the trimerized

receptor-binding domain of the spike protein of SARS-CoV-2. These types of

vaccinations containing RNA elicits a robust innate immune, such as cold flu like

symptoms. This is caused by an individual immune system, which sees the RNA as a

foreign protein. Forty-five adults ranging from ages of eighteen to fifty-five were

administrated two doses of the vaccines twenty-one days apart from one another.

These doses varied, but it was unknown who received which dose. At the end, it was

concluded that RBD-binding IgG concentrations and SARS-CoV-2 neutralizing titres in

sera increased with dose level increments and after a second dose (Mulligan et al.,

2020). It is also good to note that the cold-like symptoms that were experiment during

the first dose were also experienced in the second dose, only much stronger. The

reasoning behind this was that the immune system did not think it did a good job at

fighting off the virus the first time, so it attempts to do a better job the second time

around.

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Conclusion

General

The biological way of living cells is believed to not occur by chance. Instead,

other science such as biochemistry and biophysical aspects in life played a huge role in

the development and evolution of cells. This was done through mutations that can occur

within the process of replication and reproduction.

The first production of RNA virus is unknown, but with some look into the past, it

is believed that Neanderthal’s thousands of years ago were infected with RNA viruses.

During some sort of time spam, there was an interbreeding the occurred between these

ancient humans and the modern humans that are known today. Very little protein that

cam perform their DNA is found in the modern human. However, if there was a chance

that Neanderthals developed adaptation of viruses, they would have been passed on

just like the viruses were.

Despite the abundance amount of RNA viruses presented through the evolution

of humans, there is very little was known about these deadly viruses. This is caused by

their drastic differences that varies from virus to virus. Even so, if two viruses were

similar, chances of them staying closely related is very slim due to the high mutation

rates experienced by the instable RNA.

Other factors that contributed to this mutation rate was the genome size and

tRNA expression that occurred. There is an inversely proportional relationship between

genome size and mutation, meaning as one condition increases, the other decreases.

For example, as genome size increased, the mutation rate of the protein then sees a

decrease. Among other types of RNA, tRNA plays an important role in the reproduction

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of RNA viruses. However, an overexpression of tRNA can also lead to more

complications of diseases such as cancer and tumor growth.

Antivirals are needed to ensure that the viruses do not cause health

complications in individuals. Antivirals can come in one of two ways: the host cell

metabolism is changed, or inhibitors inhibit the interaction of the virus and the host cell.

Among components stated above and their differences, this makes antivirals hard to

accomplish. In the case with Covid-19, there was already a huge advantage that the

virus was very similar to another virus that was seen in 2002. This allows a better

equipped development of a vaccine. Despite this, it is still crucial to understand the

mechanism of other viruses to make sure that in the case of an outbreak, antivirals can

protect the wellbeing each citizen.

Future Studies

1. Using newly analytical techniques, new viruses can be detected as soon

as they are produced.

2. Studying individuals with symptoms can be a great way to understand the

mechanism of the virus itself to determine the best course of antiviral

strategies.

3. One possible course of action is blocking the receptors to limit the binding

of viruses.

4. Another possible course of action is to lower the viral load in the body. The

viral load refers to the amount of active virus that can reproduce more

virus within the organism.

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 Future studies can be done for different types of RNA viruses that function

differently depending on the genetic coding that they carry. As a result, different antiviral

strategies can be implemented depending on the type of virus. To determine this, a

virus needs to be detected within a living organism. This can be done with

immunofluorescence analysis, where the protein is strained, and its location is

identified. Understanding its mechanism can also be helpful, especially tRNA, an

important component of the coding’s journey though the living organism. This can be

done with NAIL-MS. A mechanism can help guide what harm is being done and what to

do to stop or delay it. When identifying what the mechanism is, experimental mRNA is

created in a lab to teach cells how to make that protein. The mRNA enters the living

system and makes a spike protein. Other cells in the body recognize this spike protein

as foreign and attack it, creating an immune response. This immune response produces

antibodies that help fight a real virus. This experimental study may have to be done

more than a couple of times to achieve a mRNA that targets the desired virus.

RNA viruses are always evolving and replicating within living systems whether

they are a threat or not. The purpose of these antiviral strategies is to target proteins

that contribute to the formation of viruses within the body. With new techniques being

developed to identify these viruses and learn how their mechanism works, antivirals can

address the issue before it gets out of control. As of right now, there are very few RNA

viruses that have antiviral drugs. The Covid-19 vaccine is an example. The vaccine is

not a drug that can attacks the pathogen, but much rather just inhibits the development

in the future when the virus wants to reproduce. The ideal timeline for this process is not

very ideal, as it takes it a financial burden and very time-consuming. Detecting the virus

22
is sometimes already too late in the cases where a pandemic has occurred. Needless to

say, though, this RNA vaccine has already saved many lives and allowed life to feel

more normal again. The thought of being protected brings in a feeling of comfort that

can ease the public. After all, it is better to be one step ahead of a virus.

Comment:

- Very interesting information of RNA viruses and DNA according to the recent

pandemic.

- Sources were cited thoroughly and in correct format.

- I enjoyed reading your paper even thought my background in biology is weak and

some of the contents were difficult for me to understand.

- I did not find anything needs to be corrected or changed. Great job!

Overall:

Excellent!

23
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(Figure 1 written on back page corner in pencil)

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(Figure 4 written on back page corner in pencil)

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Figure Legends

Figure 1. Seven classes of virus distinguished by genome replication and

encapsulation strategies. These classes of viruses are broken down into three

broader category of RNA viruses, DNA viruses, and reverse-transcribing viruses.

Figure 2. Factors within the Bootstrap Test. The bootstrap test is a commonly used

method that estimates a population by using dataset replacement. This allows a broader

class of resampling methods to be studied more efficiently. Proteins that interact with

the virus (VIPs) and proteins that do not interact with the virus (non-VIPs) are

considered. The results match the confounding factors between VIP and non-VIPs from

Neanderthals to both East Asian and Europeans in modern time. The red dots along the

line are the representation difference between VIPs and non-VIPs. Comparing the two,

although they are not that different, there is a noticeable difference in the conserved

density. This acknowledges the idea that Neanderthals and modern humans mixed and

passed viruses through the process of RNA and DNA over time. This could explain the

similarity, yet slight difference between Eastern Asians and Europeans. With this idea

in mind, it is possible to detect epidemics that have occurred in the past through can be

detected through host genomic adaptation.

Figure 3. Detection and quantification of green fluorescent protein (GFP) by

fluorescence-activated cell sorting (FACS). FACS is a process known to isolate

individual cells that have been infected by single virus particles with a vesicular

stomatitis virus (VSV). This process gives off a green-fluorescent protein that is easily

seen and detected. The plots show 2, 3, 4 and 5 h post-infection for BHK cells. The

denser areas are shown in red. On the contrary, the least dense areas are shown in

30
blue. They are all very similar, but the Q4 value on the bottom right of the plot is not.

These values reflect the of live cells that are infected with VSV. The values are

increasing as the plots move from left to right, meaning 5 HPI has the highest live cells

that are infected with VSV.

Figure 4. Mechanism of T-705 against influenza A virus. Influenza A virus is an RNA

virus that attacks the respiratory system. Symptoms appear to be flu-like. Influenza A

virus is a common virus and is able to cause a widespread of diseases that bring life

threatening consequences, especially if left untreated. T-705, broad spectrum

inhibitor of viral RNA polymerase and a substituted pyrazine, can help fight the virus. T-

705 is converted to its ribonucleotide (T-705 RMP). This T-705 RMP is then converted

to its 5′-triphosphate (T-705 RTP). This results in competition with Guanosine-5'-

triphosphate, inhibiting the RNA polymerase.

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