Viral Replication

Influenza virus HA attaches to sialic acid on glycoprotein or glycolipid receptors, and the virion is taken into the cell by endocytosis. At pH 5.5 within endosomes, a conformational change in the HA exposes the hydrophobic amino terminus of HA2, precipitating fusion of the viral envelope with endo-somal membrane. At the same time, protons pass into the virion through the ion channels formed by the M2 protein, dissociating Ml from the ribonucleo-

Table 31-1

Properties of Orthomyxoviruses

Three genera Influenzavtrus A,B; Influetizavirus C; unnamed Thogoto-like viruses Pleomorphic spherical or filamentous virion, diametei 80-120 nm

Envelope containing hemagglutinin (H) and neuraminidase (N) peplorners, a matrix protein (Ml)

on the inner surface, and a small number of pores composed of protein M2 Nucleocapsid of helical symmetry in 8 segments, composed of nucleoprotein (NP) and RNA

polymerase complex (PA, PB1, PB2) in association with genome Genome linear minus sense ssRNA in 8 segments, total size 13.6 kb (for influenza A and B), 7

segments (influenza C); 6 or 7 segments, 10 kb (tick-boine orthomyxoviruses) Transcription and RNA replication in the nucleus; capped 5' termini of cellular RNAs cannibalized as primers for mRNA transcnption; budding from plasma membrane Defective interfering particles and genetic reassortment frequently occur protein and releasing the nucleocapsid into the cytosol. The nucleocapsid-polymerase complex is transported into the nucleus where transcription and replication of RNA take place.

Primary transcription involves a unique phenomenon known as cap snatching: the viral endonuclease (PB2) cleaves the 5' methylguanosine cap plus about 10-13 nucleotides from heterogeneous nuclear RNA, and this is used as a primer for transcription by the viral transcriptase (PB1). Of the primary RNA transcripts so produced from the eight minus sense gene segments, six are monocistronic mRNAs which are translated directly into the proteins HA, NA, NP, PA, PB1, and PB2. The other two primary RNA transcripts undergo splicing, each yielding two mRNAs which are translated mainly in different reaching frames to produce pairs of proteins (Ml plus M2, and NS1 plus NS2) sharing only a few ammo-terminal amino acids in common.

Influenza B virus adopts another strategy to increase the coding capacity of a short genome, namely, alternative translation start sites: the bicistronic mRNA transcribed from genome segment 6 contains two nearby AUG initiation codons from which two completely different proteins, NA and NB, with distinct amino acid sequences are translated in different reading frames. Segment 7 encodes two proteins, Ml and BM2, the translation of which seems to be coupled because the BM2 initiation codon overlaps the Ml termination codon.

Replication of the eight viral RNA segments requires the synthesis of eight full-length complementary plus strands which, unlike the corresponding mRNA transcripts, must lack the 5' capped primer and the 3' poly(A) tract. Newly synthesized nucleoprotein (NP) binds to this cRNA, serving as the scaffold on which the template is copied. Later, the matrix protein Ml, which contains the zinc-finger motif characterizing proteins that bind nucleic acid, enters the nucleus and binds to the nucleoprotein-transcriptase-minus sense RNA complex, down-regulating transcription and permitting its export from the nucleus prior to assembly into virions.

The HA and NA proteins undergo glycosylation (see Fig. 3-7), polymerization, and acylation. In permissive cells HA is also subjected to cleavage into polypeptides HA] and HA2 by cellular protease(s) such as furin which recognizes a polybasic motif of arginine residues (see Chapter 7). M2 is believed to be instrumental in protecting the native HA against conformational change in the trans-Golgi vesicles by countering acidification by cellular proton pumps. HA, NA, and M2 migrate preferentially to plasma membrane on the apical surface of the cell. Ml recognizes and binds to the cytoplasmic moiety of the FIA trimer and precipitates budding (see Fig. 3-9). It is not known by what mechanism one copy of each the RNA segments is selected for incorporation into a virion. One possibility is that the eight nucleocapsid fragments are in lact loosely linked in a fixed sequence by some complex system of RNA/pro-tein recognition signals. Alternatively, the segments may be packaged at random, with the average virion enveloping substantially more than eight segments to ensure that a reasonable proportion of particles happen to contain at least one copy of each gene. Following budding, the envelope enzyme neuraminidase facilitates the release of virions by destroying sialic acid receptors on the plasma membrane which would otherwise recapture the virions and hold them at the cell surface.

Defective interfering (DI) particles, originally known as "incomplete virus," are quite often produced, especially following infection at high multiplicity (see Chapter 4).

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