Chemical Modification of Viruses

Journal of General Virology

1988

SUMMARYThe titre of defective interfering (DI) influenza virus measured by an assay based on the inhibition of cytopathology caused by A/WSN (H 1N l) influenza virus in MDCK cells was 320000-fold greater than titres measured by inhibition of infectious centre formation. Interference was less in other types of cell. By electron microscopy, we have shown that the ratio between physical particles and DI units in preparations of the DI virus was approximately unity, which suggested that one or few DI particles is/are required to confer resistance of a MDCK cell to viral cytopathology. This human DI virus interfered heterotypically with an avian H7N1 influenza virus.

mentioning

confidence: 77%

One Defective Interfering Particle per Cell Prevents Influenza Virus-mediated Cytopathology: An Efficient Assay System

McLain

Armstrong

Journal of General Virology

1988

“…Formaldehyde crosslinks by the formation of stable methylene bridges with many different groups within proteins which contain an active hydrogen [6,19]. However it has a maximum span of 2A [273.…”

Section: Crosslinkingmentioning

confidence: 99%

Chemical crosslinking of proteins of the influenza virion

Dolbear

Guest

1989

Archives of Virology

Purified influenza virus (A/FPV/Rostock/34; H7N1) was reacted with one of three chemical crosslinking reagents [dimethylsuberimidate (DMS), tartryl diazide (TDA) and formaldehyde] under conditions designed to give a ladder of crosslinked polypeptides (putative homo- and heteropolymers) when analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions. The different virion polypeptides were identified by Western blotting with monospecific antisera against HA1, HA2, NP, and M1. When reacted with any crosslinker NP preferentially formed 2mer and 4mer homopolymers while M1 formed 2mers, 4mers, 6mers, and 8mers. 2mers and 3mers of HA1 were detected after crosslinking with TDA and DMS but homopolymers of HA2 could not be identified with certainty due to comigrating M1. One heteropolymer was clearly identified as 1NP:1M1 (with DMS and TDA) and others, as expected, as components of the haemagglutinin spike 1HA1:1HA2, 2HA1:2HA2, and 3HA1:3HA2. Formaldehyde gave rise only to HA1:HA2 polymers. The presence of other heteropolymers containing NP in conjunction with HA2 and HA1 seemed likely. Whenever HA2 ran with an Mr of about 50k it comigrated with M1 suggesting it may have formed (with DMS or TDA) a 1HA2:1M1 heterodimer. However it is possible that this band consisted of HA2 homodimers comigrating with M1 homodimers. Patterns of crosslinking with DMS and TDA were similar although not identical, but those obtained with formaldehyde were markedly different. All patterns were highly reproducible.

“…Briefly virus at pH 7.6 was reacted with dimethylsuberimidate (DMS: Sigma) at 4 mg/ml for 40 min at 37 °C and the reaction terminated by the addition of Tris buffer pH 7.4. Formaldehyde reacts with active hydrogen of many different groups forming stable methylene bridges [4,8]. After neutralization with an alkaline urea buffer TDA was incubated with virus as before.…”

Section: Cross-linkingmentioning

confidence: 99%

Chemical cross-linking of proteins of the influenza virion

Dolbear²,

Guest³

1989

Archives of Virology

Purified influenza virus (A/FPV/Rostock/34;H7N1) was exposed briefly to pH 5 before returning to an alkaline pH. Virus was then reacted with one of three chemical cross-linking reagents [dimethyl suberimidate (DMS), tartryl diazide (TDA), or formaldehyde which span 11, 6, and 2A, respectively]. Cross-linked polypeptides were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and identified with monospecific antisera against HA1, HA2, NP and M1. Acidification resulted in changes in the cross-linking patterns for both HA1 and HA2 which could be detected with all three reagents. Most notable were the data with formaldehyde: under alkaline conditions cross-linking gave only HA1:HA2 heteropolymers but after brief acidification none of these were formed and in their place was a novel HA1 homodimer, an HA2 homotrimer and an HA2 of Mr 50k cross-linked to form a homodimer with another HA2 or to a heterodimer with M1. Although cross-linking by formaldehyde was much more affected by acidification of the virus than cross-linking by DMS or TDA, over half the polymers cross-linked by DMS were no longer formed after acidification. The patterns of cross-linking of NP and M1 were unchanged by low pH treatment.