# Furkan Zahid's Rabies Virus

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# Rabies Virus

* Rabies viruses belongs to rhabdoviruses family and its class is Baltimore V
* Rabies viruses has minus polarity ssRNA 11-15 kb and 180 nm long, 75 nm wide
* Hosts: Mammalians, Insects, Plants
* Caused Diseases: Rabies, Vesicular Stomatitis, Yellow Dwarf of Potato

## Entrance of the system for mammalians

**It's shape**

Source:[Science Photo Library](https://www.sciencephoto.com/media/931117/view/rabies-virus-illustration)

> Rabies virus could enter the host orgaism with using body fluids such as saliva. For this reason the mammalian organism which has rabies infection should bite the new host mammalian organism.

> Firstly; the rabies virus enter the body and it strikes towards to central nerval system to replicate them.

**Figure 2**
Source: [Research Gate](https://www.researchgate.net/figure/The-overall-pathogenesis-and-spread-of-rabies-virus-from-the-site-of-bite-to-brain-and_fig1_332143315)

> Firstly; As shown the Figure 2 the rabies virus enter the body and it strikes towards to muscles of biting site to replicate themselves initially. Then rabies viruses travels towards to central nervous system to replicate and reach the brain.

> For replication rabies viruses are used for some specific pathways to replicate themselves.

**Figure 3**
Source:[Science Direct](https://www.sciencedirect.com/science/article/pii/S0065352716300495)

* As shown in Figure 3 rabies viruses use their Spike Proteins which are set on the virus envelope to enter the host cell.
* Then Clathrin-mediated endocytosis are began to perform.

**Figure 4**
Source:[Science Direct](https://www.sciencedirect.com/science/article/pii/S0065352716300495)

* As shown in Figure 4 rabies viruses use the cytoplasmic trancription, translation, and replication.
* For this reason its own RdRp (RNA dependent RNA polymerase) is used for to constitute mRNA and leader +ssRNA from -ssRNA which belongs to virus.
**Figure 5**
Source:Molecular and Cellular Biology of Viruses Phoebe Lostroh

* As shown i Figure 5 N protein, P protein, M protein, G protein and L protein is transcribed by RdRp enzyme that belongs to virus.
* Besides as shown in Figure 5 in the EIS the long U tail is set after the transcribed sequences. This sequence also called polyA sequence which responsible for terminate the transcription.
* Thanks to these strands desired protein strands could be transcribed very accurately. Because of this multiprotein complexes does not occurs.
## Viral Proteins
* As shown in Figure 4 synthesized proteins are translated at rough endoplasmic reticulum and processed at Golgi.

### L protein

> The mononegavirus L protein, which can be as large as 250,000 Da, has
multiple domains and functions including RNA-dependent RNA polymerization, synthesis of methylated caps, and capping and polyadenylation of mRNA.

### N protein

>The other name is rabies virus nucleoprotein. This protein plays important role for manipulate the innate immunity of host organism[[1]](https://onlinelibrary.wiley.com/doi/full/10.1111/1348-0421.12058). Owing to rabies viruses could escape from the innate immunity mechanism and invasion of host cells could be easy.
>Furthermore; if innate immunity mechanisms do not perform regularly adaptive immunity systems also do not perform regularly because of lack of MHCII and MHCI complexes.
>However the first duty of this protein is constituting capsid formation of the virus.

### P protein

> P protein plays important and multiple roles during transcription and replication of the RNA genome. The multifunctional P protein is encoded by the P gene. P protein acts as a non-catalytic cofactor of large protein polymerase. It is binding to N and L protein[[2]](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548735/).

### M protein

> The M protein is an important structural component of rhabdovirus virions, forming a layer between the glycoprotein- (G-) containing outer membrane and the nucleocapsid core composed of the virus nucleoprotein (N), polymerase (L), phosphoprotein (P) and RNA genome[[3]](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2603668/).

### G protein

>The G protein is a glycoprotein which set on the outer membrane of rabies virus. G protein plays major role for enterance of Rhabdoviridae to host cell with Clathrin-mediated endocytosis[[4]](https://link.springer.com/article/10.1007/BF01314961).

## Replication mechanism

**Figure 6**
Source:Molecular and Cellular Biology of Viruses

* As shown in Figure 6 the antigenome which is synthesized for replication is used to contitute -ssRNA by L protein of Rabies virus.
**Figure 7**
Source:Molecular and Cellular Biology of Viruses Phoebe Lostroh

* As shown in Figure 7 If enough amount of -ssRNA is reached. Nuclear N proteins are synthesized to encapsulate the nucleic acid and constitute capsid formation.
* Then matrix-protein M is knitted on the capsid.

## Exit Mechanism

* The exit mechanism of rabies virus is exocytosis-based.
* The G proteins are synthesized by host cell and these proteins are set on the host cell membrane.
* At the end of capsid formation exocytosis occurs and capsid are surrounded by host cell membrane that contains G proteins on itself.

## Increased transgene expression level of rabies virus vector for transsynaptic tracing

* > Viral vectors are used for many biotechnological applications for gene therapy, targetted therapy and etc.
* > In this study, the aim is increasing of expression level of transgene which cloned on the rHEP5.0-GctL rabies virus viral vector.
* > For increasing viral vector expression level the L protein (RNA-polymerase) and M protein (Matrix protein) was focused. In this study, RV vectors were used to express mRFP as a transgene in vitro and in vivo.

### Material and Method

#### Plasmid construction and virus recovery

For increase the viral vector transcription. The L protein expression level should be increased. For this reason extra transcription units and intergenic regions are deleted between the G and L genes on the vector. Therefore G and L sequences are deleted on viral vector and new designing G and L region is inserted on the viral vector with ligation and restriction enzymes helpness.
* **Primer Strands for L:**
* Forward Primer:5’- AACCGTACGGAGACCCATATCAAGATGCTGGATCCGGGAG-3 ,
* Reverse Primer: 5’- ACAAGATCGATCTGTTGCCTTCTTTCATAGTGGTTG-3
* Forward restriction enzyme: Bsi WI
* Reverse restriction enzyme: Cla I

To construct an RV vector with attenuated M-gene expression (rHEP5.0-GML), the positions of the M gene and G gene of pHEP5.0-CVSG were exchanged, since it has been reported that viral genes located closer to the 3’ end show higher expression levels than downstream genes.

#### Results

* pHEP5.0-CVSG-mRFP, pHEP5.0-GctL-mRFP, and pHEP5.0-GML-mRFP vectors are manipulated vectors that carries mRFP protein.
* Manipulated transgene vector is rHEP5.0-GctL has best results for increasing the expression level of mRFP and To further evaluate the usefulness of this vector, RV vector is setted to expresses a slow-fluorescent timer (sFT)

**Figure 8**
Source:[NCBI](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507306/#pone.0180960.ref019)

In figure 8 transcription and expression rates of designed viral vector results are demonstrated.

According to results rHEP5.0-Gctl vector has best results. For this reason evaluate the usefulness of this vector, RV vector is setted to expresses a slow-fluorescent timer with different number of sites of sFT as shown in Figure 9

**Figure 9**
Source:[NCBI](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507306/#pone.0180960.ref019)

As shown the Figure 9 rHEP5.0-Gctl vector has high amount usefullness for transcription and expression.