|Journal of Virology Structure and Assembly|
The nucleocapsid of a negative-strand RNA virus is assembled with a single nucleocapsid protein and the viral genomic RNA. The nucleocapsid protein polymerizes along the length of the single-strand genomic RNA (viral RNA) or its cRNA. This process of encapsidation occurs concomitantly with genomic replication. Structural comparisons of several nucleocapsid-like particles show that the mechanism of RNA encapsidation in negative-strand RNA viruses has many common features. Fundamentally, there is a unifying mechanism to keep the capsid protein protomer monomeric prior to encapsidation of viral RNA. In the nucleocapsid, there is a cavity between two globular domains of the nucleocapsid protein where the viral RNA is sequestered. The viral RNA must be transiently released from the nucleocapsid in order to reveal the template RNA sequence for transcription/replication. There are cross-molecular interactions among the protein subunits linearly along the nucleocapsid to stabilize its structure. Empty capsids can form in the absence of RNA. The common characteristics of RNA encapsidation not only delineate the evolutionary relationship of negative-strand RNA viruses but also provide insights into their mechanism of replication.
IMPORTANCE What separates negative-strand RNA viruses (NSVs) from the rest of the virosphere is that the nucleocapsid of NSVs serves as the template for viral RNA synthesis. Their viral RNA-dependent RNA polymerase can induce local conformational changes in the nucleocapsid to temporarily release the RNA genome so that the viral RNA-dependent RNA polymerase can use it as the template for RNA synthesis during both transcription and replication. After RNA synthesis at the local region is completed, the viral RNA-dependent RNA polymerase processes downstream, and the RNA genome is restored in the nucleocapsid. We found that the nucleocapsid assembly of all NSVs shares three essential elements: a monomeric capsid protein protomer, parallel orientation of subunits in the linear nucleocapsid, and a (5H + 3H) motif that forms a proper cavity for sequestration of the RNA. This observation also suggests that all NSVs evolved from a common ancestor that has this unique nucleocapsid.
The 2009 H1N1 lineage represented the first detection of a novel, highly transmissible influenza A virus genotype: six gene segments originated from the North American triple-reassortant swine lineage, and two segments, NA and M, derived from the Eurasian avian-like swine lineage. As neither parental lineage transmits efficiently between humans, the adaptations and mechanisms underlying the pandemic spread of the swine-origin 2009 strain are not clear. To help identify determinants of transmission, we used reverse genetics to introduce gene segments of an early pandemic isolate, A/Netherlands/602/2009 [H1N1] (NL602), into the background of A/Puerto Rico/8/1934 [H1N1] (PR8) and evaluated the resultant viruses in a guinea pig transmission model. Whereas the NL602 virus spread efficiently, the PR8 virus did not transmit. Swapping of the HA, NA, and M segments of NL602 into the PR8 background yielded a virus with indistinguishable contact transmissibility to the wild-type pandemic strain. Consistent with earlier reports, the pandemic M segment alone accounted for much of the improvement in transmission. To aid in understanding how the M segment might affect transmission, we evaluated neuraminidase activity and virion morphology of reassortant viruses. Transmission was found to correlate with higher neuraminidase activity and a more filamentous morphology. Importantly, we found that introduction of the pandemic M segment alone resulted in an increase in the neuraminidase activity of two pairs of otherwise isogenic PR8-based viruses. Thus, our data demonstrate the surprising result that functions encoded by the influenza A virus M segment impact neuraminidase activity and, perhaps through this mechanism, have a potent effect on transmissibility.
IMPORTANCE Our work uncovers a previously unappreciated mechanism through which the influenza A virus M segment can alter the receptor-destroying activity of an influenza virus. Concomitant with changes to neuraminidase activity, the M segment impacts the morphology of the influenza A virion and transmissibility of the virus in the guinea pig model. We suggest that changes in NA activity underlie the ability of the influenza M segment to influence virus transmissibility. Furthermore, we show that coadapted M, NA, and HA segments are required to provide optimal transmissibility to an influenza virus. The M-NA functional interaction we describe appears to underlie the prominent role of the 2009 pandemic M segment in supporting efficient transmission and may be a highly important means by which influenza A viruses restore HA/NA balance following reassortment or transfer to new host environments.
pUL34 and pUL31 of herpes simplex virus (HSV) comprise the nuclear egress complex (NEC) and are required for budding at the inner nuclear membrane. pUL31 also associates with capsids, suggesting it bridges the capsid and pUL34 in the nuclear membrane to initiate budding. Previous studies showed that capsid association of pUL31 was precluded in the absence of the C terminus of pUL25, which along with pUL17 comprises the capsid vertex-specific complex, or CVSC. The present studies show that the final 20 amino acids of pUL25 are required for pUL31 capsid association. Unexpectedly, in the complete absence of pUL25, or when pUL25 capsid binding was precluded by deletion of its first 50 amino acids, pUL31 still associated with capsids. Under these conditions, pUL31 was shown to coimmunoprecipitate weakly with pUL17. Based on these data, we hypothesize that the final 20 amino acids of pUL25 are required for pUL31 to associate with capsids. In the absence of pUL25 from the capsid, regions of capsid-associated pUL17 are bound by pUL31. Immunogold electron microscopy revealed that pUL31 could associate with multiple sites on a single capsid in the nucleus of infected cells. Electron tomography revealed that immunogold particles specific to pUL31 protein bind to densities at the vertices of the capsid, a location consistent with that of the CVSC. These data suggest that pUL31 loads onto CVSCs in the nucleus to eventually bind pUL34 located within the nuclear membrane to initiate capsid budding.
IMPORTANCE This study is important because it localizes pUL31, a component previously known to be required for HSV capsids to bud through the inner nuclear membrane, to the vertex-specific complex of HSV capsids, which comprises the unique long region 25 (UL25) and UL17 gene products. It also shows this interaction is dependent on the C terminus of UL25. This information is vital for understanding how capsids bud through the inner nuclear membrane.