Understanding Influenza:Replication in the host cells

Dr. Sanjeeva Kumar M.Ph.D Research ScholarSchool of Veterinary Medicine & ScienceSutton Bonnington University ofNottingham,UK

Overview• Introduction

• Virus entry,membrane fusion and uncoating of the viral core

• Entry of vRNP into the nucleus

• Virus replication, transcription & Translation

• vRNP export

• Transport of virus proteins to the cell membrane

• Packaging and budding

• Post-translational processing

Introduction

Adaptation to new hosts & frequent antigenic alterations -challenging

Better intervention methods, A deeper understanding of the viral infectionprocess is critical

Influenza possesses an RNA genome of ~12 kilobases (kb) encodes 10–12proteins

Structurally, each RNP consists of a segment of ss-RNA, with the terminalsequences of the RNA bound by a trimeric RNA-dependent RNApolymerase and the remaining sequence bound by multiple copies ofnucleoprotein

Virus entry , Fusion and uncoatingHA is a homotrimer-spikes

Preferentially bind to sialic acid

Alpha 2,6 linkage –Human viruses

Alpha2,3 linkage –avian viruses

Swine-both

Clathrin dependent Receptor mediatedendocytosis

Edosome- low pH induces-confirmationalchange in HA0-HA2

Low pH-also induces-opening of M2 ionchannel-acidifies the viral core-releasesvRNP from M1 –into cytoplasm

Entry of vRNP into Nucleus

• The viral proteins vRNP are PA, PB1 & PB2

• Endosome to Nuclear membrane-Diffusion

• Thru classical nuclear import pathway

• NP-has two NLS signals

• NP –binds –alpha importin-beta importin- nuclear importation

• No reversal bcz of masking of NLS by M1

Virus replication & transcription• Ran-GTP binding to the importins-RNP released

• Synthesis of new RNP –replication of viral genome-transcription-translationVPencapsidation of genome- is catalysed by polymerase complex subunits (PB1,PB2& PA)

• RNA replication starts with synthesis of a +ve mRNA copy =cRNA

• Initiated by binding of PB2 to the 5’cap structure of host mRNA

• Endonuclease activity of ‘PA’ snatches the cap structure along with 10-13nucleotides along with the cap which serves as the primer for viral mRNA synthesis

• The synthesis of viral mRNA is carried out by the PB1 polymerase activity

• Transcription proceeds until the polymerase complex stalls at polyadenylationsignal near the end of the viral RNA

• Newly-synthesised vRNA is encapsidated co-transcriptionally by a viral polymeraseand free NP to form an RNP

What causes an RNP to replicate its RNArather than transcribe

• Two modes of activity differ in the source of the polymerase

• Transcribing polymerases can function in cis, copying the RNA of their RNP

• Replication, by contrast, requires a free polymerase, such as that assembled fromnewly-translated and imported viral proteins

vRNP export

• As the infection advances other viral proteins also accumulate in the nucleus. Thematrix protein M1 contains an NLS and its import into the nucleus bind to thenewly-synthesised RNP through interactions with NP

• Mediated by NEP (14 kDa) – small enough to pass thru the nuclear pore

• NEP- has two NES’s- which are recognised by exportin Crm1

• Crm1- activated for nuclear export by binding to Ran-GTP

• (Chromatin-bound Ran guanin by binding to Ran-GTP)

• Export is regulated process by various separate mechanisms-progression ofinfection triggers –apoptotic pathway-activating caspase 3 pathway

Transport of virus proteins to the cell membrane

• Nuclear export transports RNPs to the perinuclear cytoplasm

• MTOC-microtubule organising centre

• HRB-helps in dessociating from Crm1-Ran GTP complex-

• Interacts with Rab11 and transported to the apical plasma membrane

• Rab11 on RE-vasicular transport or diffusion.

Packaging and budding

• As the infection progresses, the apical plasma membrane becomes enriched withviral proteins, which together co-ordinate the budding of virions around thecomplexes of RNPs

• Glycoproteins haemagglutinin (HA) and neuraminidase (NA) span the membraneand are concentrated in lipid raft microdomains

• The membrane is also spanned by the ion channel M2, which accumulates on theboundaries of lipid rafts

• On the cytoplasmic face of the membrane, the matrix protein M1 interacts withthe cytoplasmic tails of HA, NA and M2 and with the membrane itself

• Both M1 and M2 interact with RNPs, and HA, NA, when individually over-expressed, cause the budding of virus-like particles.

Post-translational processing

• Several post-translational modifications have been described for IV proteins, Like

• Glycosylation of HA and NA

• Palmitoylation (S-acylation) of HA and M2

• SUMOylation (i.e., conjugation with the small ubiquitin like modifier) of M1 ,NS1NP PB1 and NEP/NS2

References

• “A comprehensive map of the influenza A virus replication cycle”. Yukiko Matsuoka, Hiromi Matsumae,Manami Katoh, Amie J Eisfeld, Gabriele Neumann, Takeshi Hase,Samik Ghosh, Jason E Shoemaker, Tiago JSLopes, Tokiko Watanabe, Shinji Watanabe, Satoshi Fukuyama,Hiroaki Kitano and Yoshihiro Kawaoka. BMCSystems Biology 2013, 7:97.

• “Studies on the cellular and molecular mechanisms of innate host susceptibility and resistance toinfluenza A viruses in chicken and ducks”. Kuchipudi, Suresh Varma. PhD thesis (2010).

http://theses.gla.ac.uk/2167/

• “Influenza A: Understanding the Viral Life Cycle”. Tasleem Samji. YALE JOURNAL OF BIOLOGY ANDMEDICINE 82 (2009), pp.153-159.

• “Transport of the Influenza Virus Genome from Nucleus to Nucleus”. Edward C. Hutchinson * and ErvinFodor. Viruses 2013, 5, 2424-2446.

Thanks