Rab27a is a Rab family protein involved in intracellular membrane transport. This study examined whether infection with human parainfluenza virus type 2 (hPIV-2) affects Rab27a expression levels. Using Rab-depleted and Rab-overexpressing cells, the study investigated the effects of Rab proteins on hPIV-2 growth. Results showed that depletion of Rab27a decreased hPIV-2 growth by reducing expression of the hPIV-2 F and HN proteins. This suggests Rab27a is involved in hPIV-2 growth by promoting transport of viral proteins.

1. PRESENTADO POR :
Juan Camilo Botero Guarin.
UPB-Escuela Ciencias de la salud - tercer smestre

2. INTRODUCTION
Parainfluenza
virus
• It is an ARN monocatenarian negative virus.
• It belongs to paramyxoviridae family.
• There are 4 types of parainfluenza virus that could produces pulmonary diseases like
Crup, Bronchitis, Pneumonia.
• hPIV-2 p is especially important as a pediatric respiratory pathogen, encodes a V
protein that inhibits type I interferon (IFN) induction and signaling.
Rab 27a
• Rab27a is a molecule that belongs to Rab family proteins.
• The Rab family is a member of the Ras superfamily of small
GTPases that are involved in intracellular membrane traffic .
• Each Rab protein locates on their specific mem-brane
organelle, in this case Rab27a belongs to secretory granules
with Rab26a, Rab37a.

3. INTRODUCTION
Rab 27a and
Parainfluenza
virus
• Rab27a is highly expressed in a variety of
secretory cells including endocrine, exocrine,
and immune cells, and it is also important for
outward transport in non-secretory cells
• Most of the time Rabs proteins get affect with
thoese types of ARN virus, so in this case there
is no information if Rab 27a is involved with
hPIV-2a. Human parainfluenza virus type 2 (hPIV-2)

4. GENERAL OBJECTIVE.
Here, we examined whether hPIV-2 infection affects
the expression level of these Rabs. Using Rab-
depleted and Rab-overexpressing cells, we
investigated the effects of these Rabs in hPIV-2
growth. Furthermore, we attempted to elucidate the
involvement of Rabs in the intracellular transport of
hPIV-2 components.

5. MATERIALES Y METODOS.
INMUNOBLOT
(Tambien conocida como Westernblot)
Fundamento:
tecnica que sirve para evaluar la reaccion entre un antigeno y un
anticuerpo. Prinmero se hace una electroforesis y luego esa
proteina que resulto se pone a reacionar con el anticuerpo para
realizar su debedio analisis.
Uso:
En este estudio se hizo inmunoblot para analizar los lisados celulares,
luego de preparacion y centrifugacion, de celulas Vero, separados
mediante SDS-PAGE.

6. MATERIALES Y METODOS.
RT-PCR (qRT-PCR)
Fundamento:
Es una Tecnica de amplificacion enzimatica de secuencias espeficias de
AND. Consiste en aumentar un fragmento especifico de AND usando una
Polimerasa que sinteiza grandes cantidades de AND.
Primero se aisla el DNA y luego por medio de dos primers que se unen a la
molecula de AND, se incia la secuencia.s.
Uso:
En este estudio, esta tecnica se utilizo para obtener secuencias de las
proteinas Rab en las celulas HeLa infectadas con hPIV-2, para luego poder
inducir lineas celulares con reduccion en la expresion de Rab, 8a, 27a y 11a
(tambien conocida como Reaccion en cadena de la polimerasa en tiempo real)

7. MATERIALES Y METODOS.
INMUNOFLUORESENCIA
Fundamento:
La inmunofluorescencia o IFA (Immunofluorescense Assay) es un
inmunoensayo ampliamente utilizado en el diagnóstico que utiliza
anticuerpos marcados con un fluorocromo o molécula
fluorescente que permite detectar antígenos específicos en una
sección de tejido o preparación antigénica.
Uso:
En este estudio se utilisaron los fluorocromos para identificar si las
celulas trnasformadas con pEF4/Myc-His, expresaron o no la
proteina Rab27a con etiqueta HA.

8. MATERIALES Y METODOS.
CITOMETRIA DE FLUJO
(Conocida tambien como separacion de celulas individuales)
Fundamento:
Es una Tecnica que se utiliza para el analisis celular por medio de la
idnvidualizacion celular. Este analisis se hace poniendo las celulas en un
fluido dentro de un tubo transparente, por el cual las celulas pasan una a
una sometidas rayo laser que permitira analalizar difernetes caracteristicas
( componentes, tamaño, etc…).
Uso:
En este estudio se utilizo la citometria de flujo para analizar las celulas
infectas con el virus de la parainfluenzo o con el plasmido que codificaba
para la formacion de proteinas de este mismo virus. La citometria fue
aplicada luego de haberles hecho una preparacion previa.

9. RESULTADOS.

10. RESULTADOS.
Fig.2 Effects of depletion of
RabproteinsinhPIV-2growth.Thecell lysates of
HeLa cells depleted of Rab8a (HeLa/sh-
Rab8a) (a), Rab11a (HeLa/cr-Rab11a) (c),
and Rab27a (HeLa/sh-Rab27a) (e) together
with their control cells were subjected to IB
analysis using the indi- cated Abs. Actin was
measured as a loading control.

11. RESULTADOS.

12. RESULTADOS.

13. DISCUSSION
Author CONTRIBUTION Correct or not.
(48)Stenmark H, Olkkonen VM (2001) The Rab
GTPase family. Genome Biol 2:REVIEWS3007
The Rab family is part of the Ras superfamily of
small GTPases. There are at least 60 Rab genes
in the human genome, and a number of Rab
GTPases are conserved from yeast to humans.
The different Rab GTPases are localized to the
cytosolic face of specific intracellular
membranes, where they function as regulators
of distinct steps in membrane traffic pathways.
(42) Matsumoto Y, Ohta K, Goto H, Nishio M
(2016) Parainfluenza virus chimeric mini-
replicons indicate a novel regulatory element in
the leader promoter. J Gen Virol 97:1520–1530
Describtion of The primers for each hPIV-2
mRNA and calculation hPIV-2 mRNA numbers
of copy.
(31)Fraile-Ramos A, Cepeda V, Elstak E, van der
Sluijs P (2010) Rab27a is required for human
cytomegalovirus assembly. PLoS One 5:e15318
studied the role of Rab27a, a regulator of
lysosome-related organelle transport, in HCMV
production. HCMV infection increased Rab27a
expression, and recruitment of Rab27a to
membranous structures at the assembly site.
(33)Nagashima S, Jirintai S, Takahashi M,
Kobayashi T, Nishi- zawa T, Kouki T, Yashiro T,
Okamoto H (2014) Hepatitis E virus egress
depends on the exosomal pathway, with
secretory exosomes derived from multivesicular
bodies. J Gen Virol 95:2166–2175
the introduction of a small interfering RNA
(siRNA) against Rab27A, affects the pathway of
particules virus (VHE )exocitosis. So the Rab27A
its involve in the transportation of the virus (VHE)

14. CONCLUSIONS
 The discovery of rab27a involved in the growth of hpiv-2 increasing the expression of F
and HN proteins, open the door to more researches about this relation to understand the
whole mechanism of the rab27a, and to get a really good treatment of hpiv-2.
 Molecular techniques are one of the most important inventions in recent decades, these
techniques allow the scientist to review the molecular forms to understand all the
mechanisms of infections that use diseases such as the virus. In this case, the use of these
techniques opens the general perspective on the release of viruses and allows scientists to
compare the results with other studies to recognize the unknown proteins that make us
better understand the mechanisms of all viruses, specifically hpiv-2.